US8642733B2 - T-cell stimulating protein B and methods of use - Google Patents

T-cell stimulating protein B and methods of use Download PDF

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US8642733B2
US8642733B2 US13/505,263 US201013505263A US8642733B2 US 8642733 B2 US8642733 B2 US 8642733B2 US 201013505263 A US201013505263 A US 201013505263A US 8642733 B2 US8642733 B2 US 8642733B2
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polypeptide
amino acid
tspb
acid sequence
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US20120276120A1 (en
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Gregory R. Moe
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Childrens Hospital Oakland Research Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • Neisseria meningitidis is a Gram-negative bacterium which colonizes the human upper respiratory tract and is responsible for worldwide sporadic and cyclical epidemic outbreaks of, most notably, meningitis and sepsis. The attack and morbidity rates are highest in children under 2 years of age. Like other Gram-negative bacteria, Neisseria meningitidis typically possess a cytoplasmic membrane, a peptidoglycan layer, an outer membrane which together with the capsular polysaccharide constitute the bacterial cell wall, and pili, which project into the outside environment. Encapsulated strains of Neisseria meningitidis are a major cause of bacterial meningitis and septicemia in children and young adults. The prevalence and economic importance of invasive Neisseria meningitidis infections have driven the search for effective vaccines that can confer immunity across different strains, and particularly across genetically diverse group B strains with different serotypes or serosubtypes.
  • T cell stimulating proteins were identified by Ala'Aldeen et al. in membranes of Neisseria meningitidis group B strain SD grown in iron-depleted media (Woodridge K G et al. 11 th International Pathogenic Neisseria Conference 1998, Nice, France). T cell stimulating proteins designated as TspA and TspB were subsequently identified from genomic expression libraries using rabbit antisera produced by immunizing with the membrane fractions (Woodridge K G et al, supra; Kizil G I et al. 1999 Infect. Immun 67:3533-3541). Although TspA and B were both found to stimulate T cells, they are unrelated with respect to DNA sequence.
  • TspB is homologous to Orf6, which was found to be associated with invasive meningococci when the genome sequences of commensal isolates that did not cause disease were compared with those of invasive isolates that did cause disease (Bille E J R et al. 2005 J. Exp. Med. 201:1905-1913). Inactivating Orf6 did not have an observable effect on commonly used laboratory assays for correlates of pathogenicity (e.g. growth in rabbit serum, adhesion to T84 human cells, virulence in an immunocompromised mouse model) (Bille E J R et al. 2005, supra).
  • pathogenicity e.g. growth in rabbit serum, adhesion to T84 human cells, virulence in an immunocompromised mouse model
  • TspB T-cell stimulating protein B
  • the present disclosure provides an isolated polypeptide comprising a variant peptide region (V N ) of a TspB, in which the polypeptide contains a contiguous amino acid sequence that is less than the full-length amino acid sequence of TspB.
  • FIG. 1 shows the results of the flow cytometry experiments designed to detect the presence of human or mouse IgG bound to the surface of Neisseria meningitidis group W135 strain A22 when the cells are cultured under different conditions and incubated without or with serum or purified IgG as indicated.
  • FIG. 2 shows the results of the flow cytometry experiment designed to detect the presence of human IgG bound to Neisseria meningitidis group W135 strain A22 cultured in the presence of serum from two different donors (panel A) or genetically diverse Neisseria meningitidis group W135, B, and C strains in the presence of serum from a single donor (RGM) (panel B).
  • FIG. 3 is an image of a SDS-PAGE gel stained to detect proteins purified from MenW135 strain A22 when cultured in CDM supplemented with human serum (CDM/HuS) or in Mueller-Hinton media that contain IgG-binding activity that were eluted from Protein A or Protein G beads as indicated.
  • FIG. 4 shows the amino acid sequence of the protein identified by MALDI-TOF mass fingerprinting using Mascot. Matched peptides are underlined.
  • FIG. 5 is an alignment of TspB amino acid sequences from various strains, including invasive Neisseria meningitidis group A (Z2491; NMA), group B (MC58; NMB), and group C (FAM18; NMC) and Neisseria gonorrheae (FA 1090; NGO) genomes available at NCBI retrieved using the protein sequence of NMA0776.
  • FIG. 6 shows the purification products from expressing TspB-IGB from pQE31 purified under denaturing conditions (lane A) and pET22b(+) purified under native conditions from the periplasm (lane B).
  • FIG. 7 shows an amino acid sequence of the full length TspB encoded by gene NMB1548 from the strain MC58 genome sequence.
  • FIG. 8 is an alignment of 4 of the protein sequences shown in FIG. 5 together with NMA0776.
  • the IGB domain, variant peptide region, and the proline-rich region are labeled along the lengths of the protein sequences.
  • FIG. 9 shows alignments for three groups of variant peptide sequences that share high sequence identity within each group.
  • the variant peptides are derived from amino acid sequences shown in FIG. 5 along with the amino acid sequence of NMA0776.
  • panel A is an alignment of segments of IGB domains that are N-terminal to the variant peptide regions.
  • Panel B is an alignment of segments of IGB domains that are C-terminal to the variant peptide regions. All sequences shown in FIG. 10 are derived from various TspB sequences previously shown in FIGS. 5 and 8 .
  • FIG. 11 shows binding of human IgG to wells containing either TspB1548IGB or TspB628IGB by ELISA.
  • FIG. 12 are micrographs of TspB1628IGB (panel A) and TspB1548IGB (panel B) spotted on a microscope slide and dried overnight.
  • FIG. 13 are flow cytometry experiments showing binding of various Neisseria bacteria strains to human IgG from Donor 1 and Donor 2 sera.
  • FIG. 14 are flow cytometry experiments monitoring the selection of various strains of Neisseria bacteria for the IgG-binding ability.
  • FIG. 15 shows the binding of sera from mice immunized with partially denatured TspB1628IGB to NmB strain MC58 (panel A) and to NmA strain Z2491 (panel B).
  • FIG. 16 shows binding experiments similar to FIG. 15 with additional sera from mice immunized with natively-folded TspB1548IGB (nTspB1548IGB), natively-folded TspB1628IGB (nTspB1628IGB), or partially-denatured TspB1628IGB (rfTspB1628IGB).
  • the polypeptides of the present disclosure are derived from (e.g. contain contiguous amino acid sequence of) the T-cell stimulating protein B (TspB).
  • TspB is expressed by various Neisseria strains and is also homologous to Orf6, which is found in many invasive Neisseria meningitidis strains known to cause the majority of human meningococcal diseases.
  • TspB/Orf6 expression on the cell surface of the Neisseria bacteria is enhanced when cultured in media containing human serum. It is also found that polypeptides presented herein are able to bind to human Ig independent of the antigen-specificity of the human Ig (e.g., by binding to the Fc region). Binding of TspB/Orf6 to human Ig results in activation of human complement but does not lead to productive bacteriolysis or opsonophagocytosis. Accordingly, TspB/Orf6 may facilitate the ability of the bacteria to avoid human complement activation, thus reducing bacterial clearance.
  • polypeptides of the present disclosure as a vaccine can elicit antibodies. Such antibodies may also prevent binding of TspB to human Ig. The antibodies elicited by the vaccine presented herein may then lead to the inactivation of the mechanism important for the survival of N. meningitidis in human blood.
  • polypeptides can find use in combination with other vaccines (e.g. factor H binding protein-containing vaccines, such as r3C), to enhance the bactericidal activity of an anti-Neisserial immune response elicited by the vaccine in a host.
  • other vaccines e.g. factor H binding protein-containing vaccines, such as r3C
  • Polypeptides of the present disclosure may also act as a carrier protein when conjugated to other proteins or biomolecules. Such conjugates may be used, for example, in protein-polysaccharide conjugate vaccines.
  • the present disclosure provides polypeptides derived from the amino acid sequence of TspB but not the full-length TspB, and methods of use of such polypeptides in preparation of vaccines and in eliciting antibodies (e.g. antibodies that block the function of TspB, such as binding Fc region of human IgG). Examples of embodiments of such are described below.
  • Methods to produce the subject polypeptides and/or full-length TspB are also provided herein.
  • the methods involve culturing host cells that express the polypeptides in chemically defined media supplemented with one or more components of human serum.
  • the method can also be employed to produce vesicles having the subject polypeptides and/or full-length TspB, such as microvesicles, outermembrane vesicles, or a combination of both.
  • T cell stimulating protein B (TspB), which is also known in the literature as the protein homologous to the proteins encoded by genes such as NMA0776, NMA1797, and Orf6, is a T-cell and B-cell stimulating protein.
  • Orf6 is one of the nine genes found in prophage DNA that has been linked to invasive disease caused by N. meningitidis (Bille E et al. 2005 J. Exp. Med. 201:1905-1913), particularly in young adults (Bille et al. 2008 PLoS One 3:e3885).
  • the present disclosure may use TspB or Orf6 interchangeably. See FIGS. 5 and 7 for the amino acid sequences of examples of TspB/Orf6.
  • TspB refers to a TspB that lacks the leader peptide region.
  • the leader peptide resides at the N-terminus of a naturally-occurring immature TspB.
  • the native TspB (NP — 274555) of group B strain MC58 encoded by NMB1548 is used herein as a reference sequence for purposes of residue numbering (e.g. see FIG. 7 ). It is noted, as shown in FIG. 7 , that this TspB amino acid sequence contains the leader peptide region. Because the length of TspB can differ from one gene to another by 5, 19, or up to 44 amino acid residues, the numbering system used herein to refer to an amino acid residue in a TspB protein may differ from the numbering based on the actual amino acid sequences of these proteins.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • the term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.
  • nucleic acid molecule and “polynucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • Non-limiting examples of polynucleotides include linear and circular nucleic acids, messenger RNA (mRNA), cDNA, recombinant polynucleotides, vectors, probes, and primers.
  • heterologous refers to two components that are defined by structures derived from different sources, e.g. to provide a nucleic acid or protein not found in nature.
  • heterologous is used in the context of a polypeptide, where the polypeptide includes operably linked amino acid sequences that can be derived from different polypeptides (e.g., a first component composed of a recombinant peptide and a second component derived from a native TspB polypeptide).
  • heterologous in the context of a polynucleotide encoding a chimeric polypeptide includes operably linked nucleic acid sequence that can be derived from different genes (e.g., a first component from a nucleic acid encoding a peptide according to an embodiment disclosed herein and a second component from a nucleic acid encoding a carrier polypeptide).
  • fusion polypeptides as described herein provide for presentation of epitopes in a single polypeptide that are normally found in different polypeptides.
  • heterologous nucleic acids include expression constructs in which a nucleic acid comprising a coding sequence is operably linked to a regulatory element (e.g., a promoter) that is from a genetic origin different from that of the coding sequence (e.g., to provide for expression in a host cell of interest, which may be of different genetic origin relative to the promoter, the coding sequence or both).
  • a regulatory element e.g., a promoter
  • a T7 promoter operably linked to a polynucleotide encoding a TspB polypeptide or domain thereof is said to be a heterologous nucleic acid.
  • Heterologous in the context of recombinant cells can refer to the presence of a nucleic acid (or gene product, such as a polypeptide) that is of a different genetic origin than the host cell in which it is present.
  • a Neisserial amino acid or nucleic acid sequence of one strain is heterologous to a Neisserial host of another strain.
  • operably linked refers to functional linkage between molecules to provide a desired function.
  • “operably linked” in the context of nucleic acids refers to a functional linkage between nucleic acids to provide a desired function such as transcription, translation, and the like, e.g., a functional linkage between a nucleic acid expression control sequence (such as a promoter, signal sequence, or array of transcription factor binding sites) and a second polynucleotide, wherein the expression control sequence affects transcription and/or translation of the second polynucleotide.
  • a nucleic acid expression control sequence such as a promoter, signal sequence, or array of transcription factor binding sites
  • “Operably linked” in the context of a polypeptide refers to a functional linkage between amino acid sequences (e.g., of different domains) to provide for a described activity of the polypeptide (e.g., presentation of an epitope to facilitate production of antibodies that specifically bind that epitope).
  • N-terminus and C-terminus refer to the extreme amino and carboxyl ends of the polypeptide, respectively, while “N-terminal” and “C-terminal” refer to relative positions in the amino acid sequence of the polypeptide toward the N-terminus and the C-terminus, respectively, and can include the residues at the N-terminus and C-terminus, respectively “Immediately N-terminal” or “immediately C-terminal” refers to a position of a first amino acid residue relative to a second amino acid residue where the first and second amino acid residues are covalently bound to provide a contiguous amino acid sequence.
  • “Derived from” in the context of an amino acid sequence or polynucleotide sequence is meant to indicate that the polypeptide or nucleic acid has a sequence that is based on that of a reference polypeptide or nucleic acid (e.g., a naturally occurring TspB or encoding nucleic acid), and is not meant to be limiting as to the source or method in which the protein or nucleic acid is made.
  • a reference polypeptide or nucleic acid e.g., a naturally occurring TspB or encoding nucleic acid
  • “Derived from” in the context of bacterial strains is meant to indicate that a strain was obtained through passage in vivo, or in in vitro culture, of a parental strain and/or is a recombinant cell obtained by modification of a parental strain.
  • a “TspB-derived polypeptide” is used to described polypeptides that have an amino acid sequence based on that of a naturally-occurring TspB polypeptide.
  • protective immunity means that a vaccine or immunization schedule that is administered to a mammal induces an immune response that prevents, retards the development of, or reduces the severity of a disease that is caused by Neisseria meningitidis , or diminishes or altogether eliminates the symptoms of the disease.
  • Protective immunity can be accompanied by production of bactericidal antibodies. It should be noted that production of bactericidal antibodies against Neisseria meningitidis is accepted in the field as predictive of a vaccine's protective effect in humans (Goldschneider et al. 1969 J. Exp. Med. 129:1307; Borrow et al. 2001 Infect Immun. 69:1568).
  • a disease caused by Neisseria meningitidis encompasses any clinical symptom or combination of clinical symptoms that are present in an infection of a human with Neisseria meningitidis . These symptoms include but are not limited to: colonization of the upper respiratory tract (e.g.
  • mucosa of the nasopharynx and tonsils by a pathogenic strain of Neisseria meningitidis , penetration of the bacteria into the mucosa and the submucosal vascular bed, septicemia, septic shock, inflammation, haemmorrhagic skin lesions, activation of fibrinolysis and of blood coagulation, organ dysfunction such as kidney, lung, and cardiac failure, adrenal hemorrhaging and muscular infarction, capillary leakage, edema, peripheral limb ischaemia, respiratory distress syndrome, pericarditis and meningitis.
  • broad spectrum protective immunity means that a vaccine or immunization schedule elicits “protective immunity” against at least more than one variant, subvariant, and/or strain (and can be against at least two, at least three, at least four, at least five, against at least eight, or more strains) of Neisseria meningitidis .
  • the present disclosure specifically contemplates and encompasses a vaccine and vaccination regimen that confers protection against a disease caused by a member of any capsular group (e.g., A, B, C, W135, X, Y 29E), with protection against disease caused by a capsular group B strain of Neisseria meningitidis being of interest due to the epidemiological prevalence of strains causing disease with this capsular group and lack of broadly effective group B vaccines.
  • the polypeptides of the present disclosure alone or in combination with other components present an epitope(s) of interest can be used to potentiate bactericidal antibody responses that cross-react across diverse capsular groups.
  • host or “subject” are used interchangeably herein to refer to a human and non-human animal, where non-human animals are generally referred to in the context of production of anti- Neisseria antibodies and humans are hosts of interest for vaccination to reduce the risk of disease by Neisseria infection.
  • an antibody or “specifically immunoreactive with”, in the context of an antigen (e.g., a polypeptide antigen) refers to a binding reaction which is based on and/or is probative of the presence of the antigen in a sample which may also include a heterogeneous population of other molecules.
  • an antigen e.g., a polypeptide antigen
  • the specified antibody or antibodies bind(s) to a particular antigen or antigens in a sample and do not bind in a significant amount to other molecules present in the sample.
  • an antibody or “specifically immunoreactive with” in the context of an epitope of an antigen (e.g., an epitope of a polypeptide) refers to a binding reaction which is based on and/or is probative of the presence of the epitope in an antigen (e.g., polypeptide) which may also include a heterogeneous population of other epitopes, as well as a heterogeneous population of antigens.
  • the specified antibody or antibodies bind(s) to a particular epitope of an antigen and does not bind in a significant amount to other epitopes present in the antigen and/or in the sample.
  • Immune response indicators include but are not limited to: antibody titer or specificity, as detected by an assay such as enzyme-linked immunosorbent assay (ELISA), bactericidal assay, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion; binding detection assays of, for example, spot, Western blot or antigen arrays; cytotoxicity assays, etc.
  • ELISA enzyme-linked immunosorbent assay
  • a “surface antigen” is an antigen that is present in a surface structure of a cell, particularly a bacterium such as Neisseria meningitidis (e.g. the outer membrane, capsule, pili, etc.).
  • isolated refers to a compound of interest that, if naturally occurring, is in an environment different from that in which it may naturally occur. “Isolated” is meant to include compounds that are within samples that are substantially enriched for the compound of interest and/or in which the compound of interest is partially or substantially purified. Where the compound is not naturally occurring, “isolated” indicates the compound has been separated from an environment in which it was made by either synthetic or recombinant means.
  • Enriched means that a sample is non-naturally manipulated (e.g., by an experimentalist or a clinician) so that a compound of interest is present in a greater concentration (e.g., at least a three-fold greater, at least 4-fold greater, at least 8-fold greater, at least 64-fold greater, or more) than the concentration of the compound in the starting sample, such as a biological sample (e.g., a sample in which the compound naturally occurs or in which it is present after administration), or in which the compound was made (e.g., as in a bacterial polypeptide, antibody, chimeric polypeptide, and the like).
  • a biological sample e.g., a sample in which the compound naturally occurs or in which it is present after administration
  • the compound was made e.g., as in a bacterial polypeptide, antibody, chimeric polypeptide, and the like.
  • substantially pure indicates that an entity (e.g., polypeptide) makes up greater than about 50% of the total content of the composition (e.g., total protein of the composition) and typically, greater than about 60% of the total protein content. More typically, a “substantially pure” refers to compositions in which at least 75%, at least 85%, at least 90% or more of the total composition is the entity of interest (e.g., of the total protein. Preferably, the protein will make up greater than about 90%, and more preferably, greater than about 95% of the total protein in the composition.
  • entity e.g., polypeptide
  • the present polypeptides find use in eliciting antibodies. Where the elicited antibodies bind TspB, the antibodies may inhibit binding of TspB to human Ig. Such polypeptides find use in production of immunogenic compositions which can be used in methods of eliciting anti-TspB antibodies to facilitate an anti-Neisserial immune response.
  • Polypeptides of the present disclosure contain contiguous amino acid residues derived from TspB of a length sufficient to serve as an antigenic fragment (e.g., which can be provided in an immunogenic composition).
  • Such polypeptides can have a contiguous amino acid sequence of a TspB that is at least 55 amino acid residues in length, at least 60 to 100 amino acid residues in length, at least 60 to 150 amino acid residues in length, at least 60 to 200 amino acid residues in length, at least 60 to 250 amino acid residues in length, at least 60 to 300 amino acid residues in length, or at least 60 to 400 amino acid residues in length.
  • a polypeptide of the present disclosure may also retain the activity of binding to human Ig independent of the antigen-specificity of the human Ig (e.g., by binding to the Fc region).
  • polypeptide of the present disclosure contains an immunoglobulin-binding (IGB) domain from strain NMB1548 (e.g. SEQ ID NO: 47) as demarcated in FIGS. 5 , 7 , and 8 .
  • IGB immunoglobulin-binding
  • Another example of a polypeptide of the present disclosure contains the IGB domain from another strain, such as NMB1628 (e.g. SEQ ID NO: 48), as shown in FIG. 8 .
  • IGB domain is also referred to herein as the “globular ⁇ sheet” domain or the “constant region” (CR) of TspB.
  • Full length TspB amino acid sequences are aligned in FIG. 5 , in which the IGB domains are demarcated.
  • a specific IGB domain found in Nmb 1548 TspB is demarcated in FIG. 7 .
  • IGB domains, as well as other domains of a TspB are also demarcated in FIG. 8 in examples of TspB amino acid sequences.
  • a polypeptide comprising an amino acid sequence that is substantially similar to the amino acid sequence of a Neisseria TspB includes a polypeptide comprising an amino acid sequence having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, amino acid sequence identity to a contiguous stretch of from about 55 aa to about 75 aa, from about 75 aa to about 100 aa, from about 100 aa to about 150 aa, from about 150 aa to about 200 aa, from about 200 aa to about 250 aa, from about 250 aa to about 300 aa, from about 300 aa to about 350 aa, from about 350 aa to about 400 aa, from about 400 aa to about 450 aa, from about 450 aa to about 500 aa, from about 500 aa to about 550 aa, or from about 550 aa to about 5
  • SEQ ID NO: 2 SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
  • the subject polypeptides may not contain a full-length mature TspB, and may lack at least 5, at least 10, at least 50, at least 100, up to at least 200 or more aa relative to a naturally-occurring full-length mature TspB.
  • the subject polypeptide may be a truncated TspB that lacks the leader peptide sequence and/or the N-terminal variable domain.
  • One such polypeptide can be described as a TspB encoded by gene NMB1548 which is truncated in that it lacks the 104 most N-terminal amino acid residues of the naturally-occurring immature polypeptide.
  • the leader peptide sequence and the N-terminal variable domain are sequences N-terminal to the immunoglobulin binding (IGB) domain, as shown in FIGS. 7 and 8 .
  • the polypeptides can also include or exclude the proline-rich domain.
  • the polypeptides of the present disclosure can be described as having an IGB domain as shown in FIGS. 5 , 7 , and 8 .
  • the IGB domain of TspB begins with serine 105 and ends before the proline-rich domain starting at proline 358.
  • the serine and the proline in each TspB that mark the beginning of the IGB domain and the beginning of the proline-rich domain, respectively, are bolded in FIGS. 5 and 8 .
  • the amino acid residue numbering system as referred to herein is based on NP — 274555 as encoded by NMB1548 of strain MC58.
  • the term “IGB domain” is also synonymous with the globular ⁇ sheet domain or the constant region (CR) as shown in FIG. 7 .
  • each IGB region of a TspB there is a contiguous segment of amino acid residues that do not share high amino acid sequence identity across all TspB sequences shown in FIGS. 5 and 8 .
  • This contiguous segment of amino acid sequence labeled as “variant group peptide” in FIG. 8 is flanked on both its N-terminus and the C-terminus by amino acid sequences that are highly conserved across TspB sequences shown in FIGS. 5 and 8 .
  • the amino acid sequences of the variant peptide regions can be used as a basis to categorize the variant peptide regions into groups, referred to herein as TspB groups.
  • the alignment of variant peptide regions shows highly conserved variant peptide sequences within each TspB group. Such alignments of some examples of variant peptide regions are shown in FIG. 9 .
  • V N “Variant group peptide” or “V N ” refers to a contiguous amino acid sequence of TspB which can be used to assign Neisserial strains to “groups” based on similarity of the amino acid sequence in this region. As illustrated in FIGS. 7 , 8 , and 9 , V N begins with methionine 214 (M214) and ends at lysine 277 (K277), which are shown as bolded and underlined in the amino acid sequence of TspB ( FIG. 7 ). The methionine that begins the variant group peptide region for each TspB is also bolded in FIG. 8 . As seen in FIG. 9 , variant group peptides are highly conserved within each of the three groups shown.
  • the subject polypeptide can contain one or a multimer (e.g., at least two, at least three or more) of variant group peptide regions, each independently selected from a group shown in FIG. 9 .
  • a subject polypeptide can contain a contiguous amino acid sequence of at least 84%, at least 85%, at least 86%, at least 88%, at least 89%, at least 93%, at least 95%, at least 98%, up to 100% identity with SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22, as set forth in FIG. 9 .
  • the variant group peptides may be separated by a linker.
  • V N contains three variant group peptide regions, it may be represented as V 1 -L 1 -V 2 -L 2 -V 3 .
  • each linker used is independently selected and may be different from other linkers in the subject polypeptide.
  • the polypeptide of the present disclosure can also optionally have other amino acid sequences (S N ) in the TspB, such as those within the IGB domain that flank the variant peptide region in TspB amino acid sequences shown in FIGS. 5 and 8 .
  • amino acid sequences of the IGB domain that are N-terminal to the variant peptide regions are set forth in FIG. 10 , panel A as SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and SEQ ID NO: 34.
  • amino acid sequences that are C-terminal to the variant peptide regions are set forth in FIG. 10 , panel B as SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, and SEQ ID NO: 46.
  • an example of a subject polypeptide may be represented by the following formula S 1 -L 1 -V N -L 2 -S 2 , in which L N refers to the one or more linkers which are optionally present or absent to link amino acid sequences together in the subject polypeptide, V N refers to one or more of the variant group peptide amino acid sequences as described above, and S 1 and S 2 refer to the flanking amino acid sequences (e.g. as those derived from the IGB domain), also as described above.
  • Some examples of the subject polypeptide may be represented as S 1 -V 1 -L 1 -V 2 -L 2 -V 3 -S 2 , S 1 -V 1 -S 2 -L 1 -S 1 -V 2 -S 2 , or S 1 -L 1 -V 1 -L 2 -S 2 .
  • Other combinations and permutations are also contemplated herein.
  • the subject polypeptide can contain other domains of TspB.
  • the subject polypeptide can further contain contiguous amino acid sequences derived from the proline-rich domain and/or transmembrane anchor domain, as labeled in FIGS. 7 and 8 .
  • An example of such a polypeptide may be represented by V 1 -S 1 -S 2 -S 3 , in which S 1 represents a flanking amino acid sequence derived from the IGB domain, S 2 represents the proline-rich domain, and S 3 represents the membrane anchor domain.
  • S 1 , S 2 , S 3 can also each be independently substituted with contiguous amino acid sequences from any segment of TspB or other types of proteins.
  • the polypeptide may contain a transmembrane domain from a protein other than TspB (a “heterologous” transmembrane domain).
  • the subject polypeptides containing a transmembrane domain can find use in providing the subject polypeptides in vesicles.
  • a subject polypeptide may be isolated from a natural source, e.g., is in an environment other than its naturally-occurring environment.
  • the subject polypeptide may also be recombinantly made, e.g., in a genetically modified host cell (e.g., bacteria; yeast; Pichia ; insect cells; and the like), where the genetically modified host cell is genetically modified with a nucleic acid comprising a nucleotide sequence encoding the subject polypeptide.
  • the subject polypeptide encompasses synthetic polypeptides, e.g., a subject synthetic polypeptide is synthesized chemically in a laboratory (e.g., by cell-free chemical synthesis).
  • polypeptides disclosed herein include those of the specific contiguous amino acid sequences provided herein, as well as those having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 usually no more than 10, 6, or 4 amino acid substitutions, where the substitution is usually a conservative amino acid substitution.
  • conservative amino acid substitution generally refers to substitution of amino acid residues within the following groups:
  • Conservative amino acid substitutions in the context of a peptide or polypeptide disclosed herein are selected so as to preserve either a presentation of an epitope of interest or the Fc binding activity of the polypeptide. Such presentation may be preserved by substituting with an amino acid with a side chain of similar acidity, basicity, charge, polarity, or size to the side chain of the amino acid being replaced.
  • Guidance for such substitutions may be derived on alignments of amino acid sequences of TspB. For example, according to the alignment shown in FIG. 5 , at certain residue positions that are fully conserved (*), substitution, deletion or insertion may not be allowed while at other positions where one or more residues are not conserved, an amino acid change can be tolerated. Residues that are semi-conserved (. or :) may tolerate changes that preserve charge, polarity, and/or size.
  • polypeptides of the present disclosure can be provided in a native folded form or an at least partially-denatured form (e.g., partially denatured or fully denatured form).
  • the polypeptides are at least partially denatured.
  • the polypeptides may be a mature full-length TspB or fragment thereof.
  • Partially denatured polypeptides can be used as an immunogen to elicit antibodies to TspB.
  • at least partially denatured in the context of a TspB protein or fragment thereof is meant that the protein is modified in tertiary structure relative to native TspB or fragment thereof so as to decrease the ability of the protein to form polymers with TspB or fragments thereof.
  • Partial denaturation can be accomplished by, for example, purification in the presence of a denaturant (e.g., urea, reducing agent, high salt (e.g., lithium chloride, sodium perchlorate), heating, detergent, and so forth.
  • a denaturant e.g., urea, reducing agent, high salt (e.g., lithium chloride, sodium perchlorate), heating, detergent, and so forth.
  • a denaturant e.g., urea, reducing agent, high salt (e.g., lithium chloride, sodium perchlorate), heating, detergent, and so forth.
  • partially denatured polypeptides can be obtained by purifying the polypeptides under denaturing conditions
  • At least partially denatured TspB polypeptides can be identified by examining the ability of the polypeptide to form polymers. For example, by the formation of polymers visible by microscopy or the formation of gels in aqueous solutions of TspB.
  • At least partially denatured TspB polypeptides can be identified by their ability to elicit anti-TspB antibodies that block immunoglobulin binding to native TspB. The ability of anti-TspB to block IgG binding on live Neisserial bacteria by flow cytometry.
  • Immunization with an at least partially denatured TspB or fragment thereof of the present disclosure elicits antibodies that block binding of human immunoglobulin. Such can inhibit an important mechanism for the pathogenesis of Neisseria .
  • a partially denatured polypeptide can include the IGB domain of TspB from NmB 1628 (rf TspB1628IGB), that is purified under denaturing conditions.
  • polypeptides of the present disclosure can be provided as fusion proteins containing a subject polypeptide as described herein.
  • the polypeptide as described above can be fused to an N-terminal end of another protein.
  • polypeptides of the present disclosure may contain one or more additional elements at the N- and/or C-terminus of the polypeptide, such as a protein (e.g. having an amino acid sequence heterologous to the subject polypeptide) and/or a carrier molecule.
  • additional elements such as a protein (e.g. having an amino acid sequence heterologous to the subject polypeptide) and/or a carrier molecule.
  • exemplary elements that may be linked to the subject polypeptide include a fatty acid moiety (e.g. an aliphatic carboxylic acid) and/or a carrier molecule (e.g., a carrier protein, (e.g.
  • bovine serum albumin BSA
  • ovalbumin keyhole limpet hemacyanin
  • KLH keyhole limpet hemacyanin
  • PPD purified protein derivative of tuberculin
  • a cytokine or other vaccine antigens such as fHbp, NadA, GNA2132, etc.
  • additional elements may be linked to the polypeptide via a linker, e.g. a flexible linker.
  • the polypeptide may be conjugated to a carrier molecule, e.g., to facilitate administration and/or to increase the immunogenicity in a subject to be vaccinated or treated against N. meningitidis .
  • the additional moiety may also aid in immunogenicity or forming a complex with another component in a vaccine and/or facilitate delivery to a cell or tissue of interest.
  • the polypeptide can also be modified to be conjugated to an antigen such as molecular mimetics, capsular polysaccharides, or derivatives thereof.
  • an antigen such as molecular mimetics, capsular polysaccharides, or derivatives thereof.
  • Capsular polysaccharides may be those found in meningococcal group A, C, W135, Y, and/or X. Examples of such are described in U.S. Pat. Nos. 4,727,136 and 6,030,619, the disclosures of which are incorporated herein by reference.
  • the polysaccharide derivatives may be a mixture of N-acetyl or de-N-acetyl polysialic acid derivatives, such as those containing long chain hydrocarbons, as well as aggregates thereof.
  • the polysaccharides may be attached to the subject polypeptide at either and/or both the N-/C-terminus, or to an internal amino acid residue.
  • the polysaccharides may be conjugated via a linker or directly to an amino acid residue.
  • the carrier molecules may facilitate presentation of the globular domain of TspB to the immune system and thus facilitate production of anti- N. meningitidis antibodies having properties of binding to human Ig.
  • the polypeptide can be fused at the N-terminus, fused at the C-terminus, or positioned in the scaffold such that the amino acid sequence of the globular domain of TspB is flanked by carrier protein sequence.
  • the scaffold may also facilitate display of the polypeptide on a membrane surface (e.g. a vesicle vaccine).
  • the polypeptides are heterologous to the carrier protein and thus provide for a fusion polypeptide not found in nature.
  • linkers may be optionally present in a conjugate of the subject polypeptide.
  • Linkers suitable for use in modifying the polypeptides to include additional elements exemplified above include “flexible linkers”.
  • Suitable linkers can be readily selected and can be of any of a suitable of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids.
  • Examples of flexible linkers include glycine polymers (G) n , glycine-serine polymers (including, for example, (GS) n , GSGGS n (SEQ ID NO:50) and GGGS n (SEQ ID NO:60), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers are of interest since both of these amino acids are relatively unstructured, and therefore may serve as a neutral tether between components.
  • Glycine polymers are of particular interest since glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)).
  • Exemplary flexible linkers include, but are not limited to GGSG (SEQ ID NO: 51), GGSGG (SEQ ID NO:52), GSGSG (SEQ ID NO:53), GSGGG (SEQ ID NO:54), GGGSG (SEQ ID NO:55), GSSSG (SEQ ID NO:56), and the like.
  • the ordinarily skilled artisan will recognize that design of a polypeptide conjugated to any elements described above can include linkers that are all or partially flexible, such that the linker can include a flexible linker as well as one or more portions that confer less flexible structure.
  • polypeptides of the present disclosure can be produced by any suitable method, including recombinant and non-recombinant methods (e.g., chemical synthesis). Where the polypeptide is chemically synthesized, the synthesis may proceed via liquid-phase or solid-phase. Solid-phase synthesis (SPPS) allows the incorporation of unnatural amino acids, polypeptide/protein backbone modification. Various forms of SPPS, such as Fmoc and Boc, are available for synthesizing polypeptides of the present invention. Details of the chemical synthesis are known in the art (e.g. Ganesan A. 2006 Mini Rev. Med. Chem. 6:3-10 and Camarero J A et al. 2005 Protein Pept Lett. 12:723-8).
  • SPPS Solid-phase synthesis
  • small insoluble, porous beads are treated with functional units on which polypeptide chains are built.
  • the free N-terminal amine of a solid-phase attached is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached.
  • the polypeptide remains immobilized on the solid-phase and undergoes a filtration process before being cleaved off.
  • the methods can involve any suitable construct and any suitable host cell, which can be a prokaryotic or eukaryotic cell, usually a bacterial cell (e.g. E. coli BL21(DE3) or E. coli TOP10F′) or yeast host cell.
  • a suitable host cell which can be a prokaryotic or eukaryotic cell, usually a bacterial cell (e.g. E. coli BL21(DE3) or E. coli TOP10F′) or yeast host cell.
  • Neisserial host cells used for expression of the subject polypeptides can naturally express, or be genetically modified to express other antigens of interest, such as factor H binding protein (fHbp), PorA, GNA2132, and the like.
  • a polypeptide that is expressed from a nucleic acid introduced into a host cell is referred to herein as being “exogenous”.
  • the exogenous subject polypeptide may have an amino acid sequence that is the same as, or different from, a contiguous amino acid sequence of the endogenous TspB.
  • the gene encoding the native TspB of the host cell may optionally be modified, e.g., to provide a host cell that does not express a functional endogenous TspB, such as a TspB knock out strain.
  • the host cells used may optionally have a disruption in endogenous TspB production, as described above.
  • Production methods can be used to produce outer membrane vesicles (OMV) and/or microvesicles (MV), which can be used in immunogenic compositions either alone or in combination in vesicle-based vaccines.
  • OMV outer membrane vesicles
  • MV microvesicles
  • Vesicle vaccines may be found in US Pat Pub No. 20080248065, disclosure of which is incorporated herein by reference. Exemplary host cell culture conditions are discussed below.
  • the host cell is cultured in chemically defined media supplemented with one or more components of human blood.
  • chemically defined media is intended a media in which most components used in the media are known, and thus contain no significant amounts or detectable amounts of components that are related to unidentified animal serum or unidentified complex natural sources.
  • the media in which host cells are cultured include supplements such as Cohn Fraction IV serum and/or other components of human blood.
  • Components of human blood include, but are not limited to, plasma, plasma with leukocytes (buffy coat), serum and the like. Additional components can be added alone or in various combinations to the media in order to modify one or more parameters of the screening and/or evaluating conditions.
  • Vectors can provide for extrachromosomal maintenance in a host cell or can provide for integration into the host cell genome.
  • a variety of appropriate vectors for use in production of a polypeptide of interest are available commercially (e.g. pET as used in the Example Section).
  • the expression vector provides transcriptional and translational regulatory sequences, and may provide for inducible or constitutive expression, where the coding region is operably linked under the transcriptional control of the transcriptional initiation region, and a transcriptional and translational termination region.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, leader peptide sequences (e.g.
  • the subject polypeptide can be expressed by a gene under the control of a promoter that may be heterologous to the nucleic acid encoding TspB.
  • the promoter can provide for constitutive expression (e.g. the promoter of RmpM (reduction modifiable protein), a PorA promoter, etc.), transient expression, and/or expression in the presence of human serum.
  • promoters can be either constitutive or inducible, and can be a strong constitutive promoter (e.g., T7, PorA, RmpM, and the like).
  • An example of a vector that can be used to produce the subject polypeptide is a pET plasmid as described in the examples and also known in the art.
  • the plasmid can have a pelB leader peptide, followed by nucleic acid encoding the subject polypeptide.
  • the polypeptide-encoding nucleic acid may encode any of the subject polypeptides described above (e.g. IGB, followed by proline-rich domain, which is followed by the membrane anchor domain).
  • the polypeptide-encoding nucleic acid can also followed by any tag that would facilitate protein detection and/or purification (e.g. his tag).
  • Expression constructs generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding proteins of interest.
  • a selectable marker operative in the expression host may be present to facilitate selection of cells containing the vector (e.g. erythromycin selectable marker).
  • the selectable marker may precede the promoter (e.g. a PorA promoter).
  • the expression construct may include additional elements.
  • the expression vector may have one or two replication systems, thus allowing it to be maintained in organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression construct may contain a selectable marker gene to allow the selection of transformed host cells. Selectable genes are well known in the art and will vary with the host cell used.
  • a polypeptide can be isolated from a lysate of cells genetically modified to express the polypeptide, or from a synthetic reaction mixture, by immunoaffinity purification, which generally involves contacting the sample with an anti-polypeptide antibody, washing to remove non-specifically bound material, and eluting the specifically bound polypeptide.
  • the isolated polypeptide can be further purified by dialysis and other methods normally employed in protein purification methods.
  • the polypeptide may be isolated using metal chelate chromatography methods.
  • the subject polypeptides may be prepared in substantially pure or isolated form (e.g., free from other polypeptides).
  • the polypeptide is present in a composition that is enriched for the polypeptide relative to other components that may be present (e.g., other polypeptides or other host cell components).
  • Purified polypeptide may be provided such that the polypeptide is present in a composition that is substantially free of other expressed polypeptides, e.g., less than 90%, usually less than 60% and more usually less than 50% of the composition is made up of other expressed polypeptides.
  • coupling may be achieved using a bifunctional coupling agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), carbodiimide, glutaraldehyde, succinic anhydride, and the like.
  • a bifunctional coupling agent such as maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), carbodiimide, glutaraldehyde, succinic anhydride, and the like.
  • the antigen and carrier protein may be generated as a fusion protein, described above.
  • the subject polypeptide may be generated using recombinant techniques to manipulate nucleic acids of different TspB known in the art to provide constructs encoding a polypeptide of interest. It will be appreciated that provided an amino acid sequence, the ordinarily skilled artisan will immediately recognize a variety of different nucleic acids encoding such amino acid sequence in view of the knowledge of the genetic code.
  • TspB polypeptides For production of subject polypeptides derived from naturally-occurring polypeptides, it is noted that nucleic acids encoding a variety of different TspBs of Neisseria bacteria are known and available in the art. Examples of TspB polypeptides and their nucleic acids are described in, for example, U.S. Pat. No. 6,861,507; Robinson K et al. 2005 Infect. and Immun. 73:4684-4692; Bille E et al. 2005 J. Exp. Med. 201:1905-1913; and Bille E et al. 2008 PLoS One 3:e3885, disclosures of which are incorporated here by reference.
  • Nucleic acid (and amino acid sequences) for various TspB are also provided in GenBank as accession nos., such as: Gene ID: NMA0776, protein access. no. A1IQJ2 (from N. meningitidis serogroup A); Gene ID: NMA1173, protein access. no. A1IRJ0 (from N. meningitidis serogroup A); Gene ID: NMA1797, protein access. no. A1IT08 (from N. meningitidis serogroup A); Gene ID: NMA2005, protein access. no. A1ITJ3 (from N. meningitidis serogroup A); Gene ID: NMC0025, protein access. no. A1KR75 (from N.
  • A1KVX5 (from N. meningitidis serogroup C, serotype 2a); Gene ID: NMCC — 0151, protein access. no. A9M0A8 (from N. meningitidis serogroup C); Gene ID: NMCC — 0919, protein access. no. A9M243 (from N. meningitidis serogroup C); Gene ID: NGK — 1481, protein access. no. B4RJA5 (from N. gonorrhoeae ); Gene ID: NGK — 2027, protein access. no. B4RNK3 (from N. gonorrhoeae ); Gene ID: NGO1140, protein access. no.
  • Q5F7B3 (from N. gonorrhoeae ); Gene ID: NGO1167, protein access. no. Q5F7K4 (from N. gonorrhoeae ); Gene ID: NMB0480, protein access. no. Q7DDP6 (from N. meningitidis serogroup B); Gene ID: NMB1747, protein access. no. Q9JY49 (from N. meningitidis serogroup B); Gene ID: NMB1628, protein access. no. Q9JYD9 (from N. meningitidis serogroup B); and Gene ID: NMB1548, protein access. no. Q9JYK0 (from N. meningitidis serogroup B).
  • nucleotide sequences encoding the subject polypeptide may be modified so as to optimize the codon usage to facilitate expression in a host cell of interest (e.g., E. coli, N. meningitidis , human (as in the case of a DNA-based vaccine), and the like). Methods for production of codon optimized sequences are known in the art.
  • the nucleic acid sequences may also be modified to express any of the polypeptide of interest described above. Examples include a TspB with both the leader peptide sequence and the N-terminal variable domain truncated, or a segment of the TspB containing the IGB domain.
  • nucleic acids are used in host cells to provide polypeptides in a vesicle (e.g. vesicle-based vaccine)
  • the nucleic acids may also encode a full-length mature TspB, any subject polypeptides described above, or a combination of both.
  • Antigen composition “antigenic composition” or “immunogenic composition” is used herein as a matter of convenience to refer generically to compositions comprising a polypeptide that binds to Fc region of human Ig as disclosed herein, in which the subject polypeptide may be optionally conjugated and/or provided in combination to enhance immunogenicity.
  • Compositions useful for eliciting anti- N. meningitidis antibodies in a human are specifically contemplated by the present disclosure.
  • Antigenic compositions can contain 2, 3, 4, 5, 6 or more different polypeptides as described herein, where each subject polypeptide may differ in amino acid sequence.
  • polypeptide antigens that can elicit anti-Neisserial antibodies in addition to or other than TspB may be optionally included in the subject composition.
  • the polypeptides can be provided in combination with polypeptides comprising amino acid sequences of a v.1, v.2, and/or v.3 factor H binding protein (fHbp), e.g., to provide for production of antibodies that bind other proteins associated with the Neisserial diseases, and the like.
  • fHbp v.1, v.2, and/or v.3 factor H binding protein
  • the subject polypeptides are administered in combination with (i.e., in the same or different formulations) with a composition including one or more of the Neisserial antigens fHbp, GNA2132, NadA, GNA2091 and GNA1030, or an antigenic fragment or fusion protein thereof.
  • a composition including one or more of the Neisserial antigens fHbp, GNA2132, NadA, GNA2091 and GNA1030, or an antigenic fragment or fusion protein thereof.
  • Exemplary combinations include fHbp, GNA2132 and NadA; and fHbp, GNA2132, Nad A, GNA2091 and GNA1030.
  • the polypeptide of the present disclosure is provided in combination with the 5 component recombinant protein vaccine as described in Giuliani et al. 2006 Proc Natl Acad Sci USA 103:10834-9.
  • the subject polypeptides can be provided in combination with any of a variety of compositions containing N. meningitidis vesicles (e.g., microvesicles and/or outer membrane vesicles), which are produced from an N. meningitidis strain expressing fHbp, particularly a v.1 and/or v.2 fHbp and/or v.3 fHbp.
  • N. meningitidis vesicles e.g., microvesicles and/or outer membrane vesicles
  • fHbp e.g., a v.1 and/or v.2 fHbp and/or v.3 fHbp.
  • Antigenic compositions generally comprise an immunologically effective amount of a subject polypeptide, and may further include other compatible components, as may be desired.
  • immunologically effective amount is meant that the administration of that amount to an individual, either in a single dose, or as part of a series of the same or different antigenic compositions, is effective to elicit and/or potentiate an antibody response effective for treatment or prevention of a symptom of, or disease caused by, for example, infection by Neisseria , particularly N. meningitidis .
  • This amount varies depending upon the health and physical condition of the individual to be treated, age, the capacity of the individual's immune system to produce antibodies, the degree of protection desired, the formulation of the vaccine, the treating clinician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • concentration of the subject polypeptides in a formulation can vary widely (e.g., from less than about 0.1%, usually at or at least about 2% to as much as 20% to 50% or more by weight) and will usually be selected primarily based on fluid volumes, viscosities, and patient-based factors in accordance with the particular mode of administration selected and the patient's needs.
  • Polypeptide compositions can be provided in a pharmaceutically acceptable excipient, which can be a solution such as a sterile aqueous solution, often a saline solution, or they can be provided in powder form. Such excipients can be substantially inert, if desired.
  • the polypeptide compositions can include an adjuvant.
  • suitable adjuvants that can be used in humans include, but are not necessarily limited to, alum, aluminum phosphate, aluminum hydroxide, MF59 (4.3% weight/volume squalene, 0.5% weight/volume Tween 80TM, 0.5% weight/volume Span 85), CpG-containing nucleic acid (where the cytosine is unmethylated), QS21, MPL, 3DMPL, extracts from Aquilla, ISCOMS, LT/CT mutants, poly(D,L-lactide-co-glycolide) (PLG) microparticles, Quil A, interleukins, and the like.
  • thr-MDP N-acetyl-muramyl-L-threonyl-D-isoglutamine
  • CGP 11637 N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine
  • nor-MDP N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dip-almitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine
  • CGP 19835A referred to as MTP-PE
  • RIBI which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween 80 emulsion.
  • the effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against
  • adjuvants to enhance effectiveness of the composition include, but are not limited to: (1) oil-in-water emulsion formulations (with or without other specific immunostimulating agents such as muramyl peptides (see below) or bacterial cell wall components), such as for example (a) MF59TM (WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds.
  • Span 85 (optionally containing MTP-PE) formulated into submicron particles using a microfluidizer, (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) RIBITM adjuvant system (RAS), (Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components such as monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (DetoxTM); (2) saponin adjuvants, such as QS21 or StimulonTM (Cambridge
  • interferons e.g. gamma interferon
  • M-CSF macrophage colony stimulating factor
  • TNF tumor necrosis factor
  • MPL monophosphoryl lipid A
  • 3dMPL 3-O-deacylated MPL
  • WO 00/56358 combinations of 3dMPL with, for example, QS21 and/or oil-in-water emulsions e.g. EP-A-0835318, EP-A-0735898, EP-A-0761231; (7) oligonucleotides comprising CpG motifs (Krieg Vaccine 2000, 19, 618-622; Krieg Curr opin Mol Ther 2001 3:15-24; Roman et al., Nat. Med, 1997, 3, 849-854; Weiner et al., PNAS USA, 1997, 94, 10833-10837; Davis et al, J. Immunol, 1998, 160, 810-876; Chu et al., J. Exp.
  • a polyoxyethylene ether or a polyoxyethylene ester e.g WO 99/52549
  • a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol WO 01/21207
  • a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol (WO 01/21152);
  • a saponin and an immunostimulatory oligonucleotide e.g.
  • a CpG oligonucleotide (WO 00/62800); (11) an immunostimulant and a particle of metal salt e.g WO 00/23105; (12) a saponin and an oil-in-water emulsion e.g. WO 99/11241; (13) a saponin (e.g QS21)+3dMPL+IM2 (optionally+a sterol) e.g WO 98/57659; (14) other substances that act as immunostimulating agents to enhance the efficacy of the composition.
  • Muramyl peptides include N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutarninyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE), etc.
  • Adjuvants suitable for administration to a human are of particular interest.
  • the polypeptide compositions may comprise other components, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • the polypeptide-containing formulations can be provided in the form of a solution, suspension, tablet, pill, capsule, powder, gel, cream, lotion, ointment, aerosol or the like. It is recognized that oral administration can require protection of the compositions from digestion. This can be accomplished either by association of the composition with an agent that renders it resistant to acidic and enzymatic hydrolysis or by packaging the composition in an appropriately resistant carrier molecule. Means of protecting from digestion are well known in the art.
  • the polypeptide-containing formulations may be provided so as to enhance serum half-life of the subject polypeptide following administration.
  • the polypeptide may be provided in a liposome formulation, prepared as a colloid, or other conventional techniques for extending serum half-life.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. 1980 Ann. Rev. Biophys. Bioeng. 9:467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.
  • the preparations may also be provided in controlled release or slow-release forms.
  • compositions for use in eliciting an immune response against N. meningitidis in a subject.
  • “Combination” as used herein is meant to include compositions that are formulated separately for separate administration (e.g., as may be provided in a kit), as well as for administration in a single formulation (i.e., “co-formulated”).
  • exemplary antigenic compositions include one or more of the polypeptides of the present disclosure together with recombinant vaccines that include a different Neisserial protein or fragment thereof may be provided as an isolated protein and/or may be provided in a vesicle vaccine.
  • the various components described herein that may be included in an antigenic composition of the present disclosure may be combined together in vitro, ex vivo, and/or be already provided by the host cells used in the subject method.
  • One of ordinary skill in the art may make the combination using any method of choice.
  • the antigenic compositions can include elements described above that can be fused to the polypeptides of the present disclosure or as a separate component in the composition.
  • An example of a component that may be included in the composition in addition to the subject polypeptides is the “r3C” vaccine, which contains a recombinant fHbp v.1 (encoded by gene from strain MC58), GNA2132 (encoded by gene from strain NZ98/254), and NadA (encoded by the gene from strain 2996).
  • the vesicles may be combined with the 5 component recombinant protein vaccine (5C or rSCV).
  • the 5 component recombinant protein vaccine refers to a recombinant protein vaccine containing GNA2091 fused with fHbp v. 1, GNA2132 (from NZ98/254) fused with GNA1030, and NadA and is described in Giuliani et al. 2006 Proc Natl Acad Sci USA 103:10834-9.
  • the polypeptides described herein can be generally administered in an immunogenic composition to a human subject that is at risk of acquiring a Neisserial disease so as to prevent or at least partially arrest the development of disease and its complications.
  • An amount adequate to accomplish this is defined as a “therapeutically effective dose.” Amounts effective for use will depend on, e.g., the immunogenic composition, the manner of administration, the weight and general state of health of the patient, and the judgment of the prescribing physician. Single or multiple doses of the immunogenic compositions may be administered depending on the dosage and frequency required and tolerated by the patient, and route of administration.
  • the polypeptides described herein are generally administered in an amount effective to elicit a humoral immune response in the host.
  • amounts for immunization will vary, and can generally range from about 1 ⁇ g to 100 ⁇ g per 70 kg patient, usually 5 ⁇ g to 50 ⁇ g/70 kg.
  • Substantially higher dosages e.g. 10 mg to 100 mg or more
  • the initial administration can be followed by booster immunization of the same of different polypeptide-containing antigenic composition.
  • vaccination involves at least one booster, more usually two boosters.
  • immunization can be accomplished by administration by any suitable route, including administration of the composition orally, nasally, nasopharyngeally, enterically, topically, transdermally, subcutaneously, intramuscularly, in tablet, solid, powdered, liquid, aerosol form, locally or systemically, with or without added excipients as may be suitable for the desired route of administration.
  • suitable routes including administration of the composition orally, nasally, nasopharyngeally, enterically, topically, transdermally, subcutaneously, intramuscularly, in tablet, solid, powdered, liquid, aerosol form, locally or systemically, with or without added excipients as may be suitable for the desired route of administration.
  • Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa. (1980).
  • An immune response can be assessed by known methods (e.g. by obtaining serum from the individual before and after the initial immunization, and demonstrating a change in the individual's immune status, for example an immunoprecipitation assay, or an ELISA, or a bactericidal assay, or a Western blot, or flow cytometric assay, or the like).
  • known methods e.g. by obtaining serum from the individual before and after the initial immunization, and demonstrating a change in the individual's immune status, for example an immunoprecipitation assay, or an ELISA, or a bactericidal assay, or a Western blot, or flow cytometric assay, or the like).
  • the antigenic compositions can be administered to a human subject, which subject may be immunologically naive with respect to Neisseria meningitidis .
  • the subject is a human child about five years or younger, and preferably about two years old or younger, and the antigenic compositions are administered at any one or more of the following times: two weeks, one month, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 months, or one year or 15, 18, or 21 months after birth, or at 2, 3, 4, or 5 years of age.
  • the present disclosure also features methods of screening for effective vaccines, and screening for candidate agents (e.g. antibodies), nucleic acid encoding the same, or immunogens effective for eliciting antisera.
  • candidate agents e.g. antibodies
  • nucleic acid encoding the same e.g. antibodies
  • immunogens effective for eliciting antisera e.g. antibodies
  • the methods can be used to assess antibody binding, both in terms of via the Fc portion of an antibody and binding of the antigen-specific portion (i.e, the ability of the antibody to bind to TspB through an antigen-binding region).
  • Such assay methods find use in screening candidate agents which can inhibit binding of TspB to human Ig, in which the binding is independent of the antigen-specificity (e.g., by binding to the Fc region).
  • Methods can encompass screening antibodies to identify those that inhibit the function of a Neisserial protein (e.g. binding of TspB to human IgG).
  • a human Ig or fragment thereof e.g. Fc portion
  • the subject method involves: analyzing a candidate agent (e.g., an antibody) that may be a candidate for inhibiting binding of TspB to human Ig, in which the binding is independent of the antigen specificity of the Ig (e.g. by binding in the Fc region).
  • a candidate agent e.g., an antibody
  • the method may involve immunizing a non-human animal with a vaccine (e.g., a polypeptide containing an epitope of TspB), collecting serum antibodies from such immunized animals, and screening the serum for the ability to inhibit TspB binding to Ig.
  • a vaccine e.g., a polypeptide containing an epitope of TspB
  • candidate agent as used herein describes any molecule, e.g. protein or small molecule compound. Accordingly, candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons.
  • candidate agents include functional groups to facilitate structural interaction with proteins, e.g., hydrogen bonding, and can include an amine, carbonyl, hydroxyl, or carboxyl group.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including, but not limited to: peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate agents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • analysis of candidate agents may be carried out by assessing the ability of TspB to bind to human Ig or a fragment thereof (e.g. Fc fragment) in the presence of a candidate agent, either in a cell-free or cell-based assay.
  • Serial dilution experiments can be carried out to select for candidates that exhibit many fold increase of affinity to TspB relative to human Ig or a fragment thereof (e.g. Fc fragment).
  • the TspB used in the assay may include full-length or fragment thereof, isolated from Neisserial bacteria, synthetically made, recombinantly made, and/or present on the cell surface of Neisserial bacteria.
  • the candidate agent may also be assessed for its ability to facilitate bactericidal activity using methods known in the art and described in the Examples below.
  • a polypeptide containing human Ig (or fragment thereof), the TspB protein, and/or the subject polypeptides may be labeled in the subject screening method.
  • the TspB, the subject polypeptides, and/or a polypeptide containing human Ig or a fragment thereof (e.g. Fc portion) can optionally be immobilized on a support through covalent or non-covalent attachment.
  • An exemplary screening assay may be a competitive binding assay, in which a polypeptide containing a human Ig or a fragment thereof (e.g. Fc region) can be detectably labeled and bound to immobilized TspB. Displacement of this detectably labeled reagent from the bound TspB is then assessed in the presence of the candidate agent.
  • Detectable label includes those that provide for directly detectable signal (e.g., fluorophore) or indirectly detectable signal (e.g., use of a secondary antibody having a detectable label).
  • a radioactive label e.g., a fluorescent label, chemiluminescers, enzymes, specific binding molecules, particles, e.g. magnetic particles, and the like. Additional elements can be provided to facilitate isolation (e.g., biotin tag, an HA tag, a poly-Histidine tag, an immunologically detectable tag) through various methods (e.g., affinity capture, etc.).
  • Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc.
  • the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.
  • the candidate agent may be also assessed for its affinity for a bacterial cell of interest.
  • Bacterial cells for screening can be selected from Escherichia coli K1, Escherichia coli K92, any Neisserial bacteria (e.g. Neisseria meningitidis Serogroup B, Neisseria meningitidis Serogroup C, Neisseria meningitidis Serogroup X, Neisseria meningitidis Serogroup Y, Neisseria meningitidis Serogroup W-135, and the like).
  • the method can involve isolating one or more antibodies from the antibody population that bind bacterial cell, bind TspB, and/or inhibit binding of TspB to human Ig.
  • the isolated antibodies identified by such screening can facilitate bactericidal activity against the bacterial cell, e.g., by facilitating complement-mediated bactericidal activity and/or opsonophagocytotic activity capable of decreasing the viability of the bacteria in human blood.
  • the bacterial cell is cultured in chemically defined media supplemented with one or more components of human blood.
  • Chemically defined media that are supplemented with human serum are employed in the subject screening methods for conditions under which the bacterial cells are cultured and/or for conditions under which candidate agent binding (e.g. antibody binding) is performed. Accordingly, the screening methods of the present disclosure is carried out under conditions that simulate bacteremia, and/or neoplasia in humans.
  • “Chemically defined media” as used in connection with the subject screening assays described herein is intended to be a media that is deficient of non-human animal byproducts.
  • the chemically defined media for Neisseria meningitidis employed in the present disclosure contain known components suitable for expression of the desired antigenic determinant, is deficient of non-human animal byproducts, and is further supplemented with one or more components of human blood.
  • Components of human blood include but are not limited to plasma, plasma with leukocytes, complement, clotting factors such fibrinogen, fibronectin, fibrin, plasminogen, ⁇ -globulins, ⁇ -globulins, and the like.
  • the plasma is heat-inactivated in order to inactivate intrinsic complement activity.
  • the assay involves detecting binding to a particular human antibody, the plasma is depleted of that particular antibody or class of antibody (e.g., IgG).
  • the plasma is also obtained from a donor that lacks intrinsic bacteriocidal activity against the N. meningitidis cell of interest.
  • an example of human blood component is human Cohn Fraction IV serum, which can be obtained from commercial sources.
  • the screening and/or evaluating may further include the step of: comparing binding of a candidate agent (e.g. antibody) to a bacterial cell grown in an undefined media, such as a media containing one or more non-human animal byproducts, such as bovine serum and the like.
  • a candidate agent e.g. antibody
  • an undefined media such as a media containing one or more non-human animal byproducts, such as bovine serum and the like.
  • undefined media suitable for this purpose are known.
  • examples of undefined media for supporting growth and/or viability of a Neisseria meningitidis cell include, but are not limited to, Mueller-Hinton media and the like.
  • the order of steps of the subject methods in which comparing agent binding to a bacterial cell cultured in chemically defined and/or undefined media can be varied, and thus chosen by the user for a given end use.
  • the present disclosure also provides isolated antibody produced by the above methods, as well as an isolated cell line capable of producing an isolated antibody produced according to the method. Isolation and production in a cell line can be carried out by any number of procedures known in the art.
  • the present disclosure further provides methods to screen candidate agents (e.g. antibodies) effective in inhibiting TspB Ig binding by detecting the effect of the candidate agent on formation of polymeric structures of TspB (e.g. IGB domain thereof, such as that of NmB 1628).
  • candidate agents e.g. antibodies
  • the antibodies may be elicited by administration of candidate antigen.
  • the methods make use of the tendency of TspB polypeptides to aggregate into polymeric structures and their ability to be dispersed when contacted with antibodies that inhibit Ig-binding activity of TspB.
  • the methods involve contacting a candidate agent (e.g.
  • Detection of polymeric structures can be accomplished by any suitable means, e.g., detection of aggregates in a reaction mixture using a light microscope.
  • the reaction mixture can be provided in an aqueous solution and/or in a gel.
  • the candidate agent disperses and/or inhibits aggregation of the present polypeptides relative to aggregation in the absence of the candidate agent, the candidate agent is identified as a candidate for inhibition of Ig-binding activity of TspB.
  • kits for using the compositions disclosed herein and for practicing the methods, as described above may be provided for administration of a vaccine (e.g., prophylactic or therapeutic) against N. meningitidis .
  • the kit can include one or more of the polypeptides disclosed herein or encoding-nucleic acids, which may be provided in a sterile container, and can be provided in formulation with a pharmaceutically acceptable excipient for administration to a subject.
  • the polypeptides can be provided with a formulation of an anti- N.
  • meningitidis vaccine that provides for production of anti-TspB antibodies (e.g., a formulation containing recombinant fragment of TspB, vesicles from a TspB-expressing strain cultured with human serum, and the like), where the polypeptides of the present disclosure may be formulated separately or in combination with such vaccine.
  • anti-TspB antibodies e.g., a formulation containing recombinant fragment of TspB, vesicles from a TspB-expressing strain cultured with human serum, and the like
  • the polypeptides of the present disclosure may be formulated separately or in combination with such vaccine.
  • kits can further include instructions for using the components of the kit to practice the subject methods.
  • the instructions for practicing the subject methods are generally recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or subpackaging), etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g. CD-ROM, diskette, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g. via the internet, are provided.
  • An example of this embodiment is a kit that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, this means for obtaining the instructions is recorded on a suitable substrate.
  • the composition of the Catlin 6 media was adapted from Fossa da Paz (Fossa da Paz et al. 2003 Brazil. J. Micro. 34:27-32). Media was prepared fresh prior to each experiment by combining pre-made stock solutions of the amino acids, salts, glucose and iron.
  • the CDM was supplemented with five percent IgG depleted sera that was heat inactivated at 56° C. (CDM-dHuS) prepared as described below. It was observed that adding 2M glutamine (UCSF cell culture facility) decreased the doubling time of some Nm strains cultured in CDM and was, therefore, included in the preparation of CDM. Cysteine was excluded from the Catlin 6 formulation because it was found that Catlin media without cysteine resulted in more consistent growth rates for most Nm strains than media with cysteine.
  • a 5 ml Protein G column (HiTrap Protein G HP, GE Healthcare Bio-sciences, Piscataway N.J.) was fitted with sterilized luer lock valves and two 10 ml syringes on the top and bottom of the column 25 ml of sterile filtered Dulbeccos buffered saline with Ca+ and Mg+, 0.5% glucose (DPBS+glu) was used to wash the column which was then chilled for 20 minutes at 4° C. Serum was removed from ⁇ 80° C. storage and thawed on ice. 5.5 mls of serum was loaded onto the column and was placed at 4° C. for 5 minutes. DPBS+glu was then applied to the column to displace IgG depleted serum.
  • Meningococcal strains (group W135 strain A22, group B strain NMB, group C strain 4243) were grown to an OD 620nm of ⁇ 0.6 in Mueller-Hinton (MH; BD) media containing 0.25% (weight/volume) glucose, Catlin 6 (CDM) media (9), or Catlin 6 media supplemented with 5% (volume/volume) human (HuS) or mouse serum (MoS).
  • MH Mueller-Hinton
  • CDM Catlin 6
  • Human human
  • MoS mouse serum
  • the serum from human donors was heat inactivated by incubating the serum at 56° C. for 30 minutes (HuS) and depleted of IgG (dHuS) by passing the serum over a Protein G column as described above.
  • the cells were pelleted, washed, and resuspended in 80% of the original volume in blocking buffer (PBS buffer containing 1% bovine serum albumin (Sigma)). The mixture of cells was incubated at 4° C. for 1 hr with periodic gentle agitation. The cells were pelleted and resuspended in 200 ⁇ l of a 1:200 dilution in blocking buffer of fluorescein isothiocyanate (FITC)-conjugated goat anti-human secondary antibodies. FITC-conjugated antibodies against IgG(H+L) F(ab′) 2 and IgM (Jackson ImmunoResearch, West Grove, Pa.).
  • FITC fluorescein isothiocyanate
  • the tubes were incubated for 1 hr at 4° C. with periodic gentle agitation.
  • the cells were pelleted and resuspended in 400 ⁇ l of PBS containing 0.5% formaldehyde (weight/volume), freshly made and filtered (Steriflip, Millipore, Billerica, Mass.).
  • the samples were immediately analyzed by flow cytometry (BD FACSCalibur System, BD Biosciences, San Jose, Calif.).
  • TspB-IGB vaccine i.e. TspB-IGB vaccine
  • 50% saline/50% Freund's complete adjuvant (Pierce) emulsion or 3.25 mg/ml alhydrogel (alum) (Brenntag Biosector, Frederikssund, Denmark) or saline alone by ip injection.
  • Blood samples were obtained by lancet of the facial vein 40 days after each injection and tested by ELISA.
  • Booster doses were given at post 28 days with incomplete Freund's adjuvant (Pierce), alum or saline alone and titers of antisera obtained 14 days post immunization were evaluated.
  • Antisera titers were determined by ELISA. The antisera from individual mice were pooled and all further experiments were done with the pooled antisera.
  • ELISA plates are were prepared by diluting each TspB-IGB 1:200 to 10 ⁇ g/ml in PBS and adding 100 ⁇ l per well to a 96-well microtiter plate (Immulon II HB). The plates were stored overnight at 4° C. before use.
  • the plates were washed with PBS buffer 5 times and blocked with PBS buffer containing 1% (weight/volume) of BSA (Blocking buffer) for one hour at ambient temperature.
  • the antisera were added in Blocking buffer at 1:100 dilution, followed by serial 3-fold dilutions (in duplicate). After overnight incubation at 4° C., the plates were washed with PBS buffer 5 times and rabbit anti-mouse-alkaline phosphatase conjugate antibody (Zymed) diluted 1:3000 in Blocking buffer was added.
  • the plates were washed (5 ⁇ ) with PBS buffer and the bound antibody was detected by adding 1 mg/ml p-nitrophenyl phosphate substrate (Sigma-Aldrich) in 50 mM sodium carbonate buffer, pH 9, containing 1 mM MgCl 2 .
  • the absorbance at 405 nm after 30 minutes incubation at ambient temperature was measured using a Molecular Devices SpectraMax 340 microtiter plate reader. Antisera were tested against the TspB-IGB.
  • Frozen cell pellets from 250 ml of cell culture were thawed and resuspended in 0.5% (weight/volume) octyl glucopyranoside (Calbiochem, San Diego, Calif.) dissolved in 20 ml of 50 mM Tris.HCl, 150 mM sodium chloride, pH 7.5 (TBS) and 20 ml of PBS containing 0.1 mM phenylmethanesulfonyl fluoride (PMSF) and 10 ⁇ g/ml DNAse (Sigma-Aldrich). The mixture was homogenized using a syringe fitted with an 18 G needle and a length of Teflon tubing by repeatedly forcing the solution back and forth through the needle.
  • octyl glucopyranoside Calbiochem, San Diego, Calif.
  • the homogenized solution was agitated at ambient temperature on a rocking platform for 20 min then centrifuged at 20,000 ⁇ g for 10 minutes. The supernatant was discarded and the pellet was suspended in 0.5% (volume/volume) EmpigenTT (Calbiochem) dissolved in 10 ml of TBS and 10 ml of PBS containing 0.1 mM PMSF and 10 ⁇ g/ml DNAse.
  • the homogenized solution was agitated at ambient temperature on a rocking platform for 20 min then centrifuged as above. The resulting supernatant was transferred to new tube, while the pellet was extracted a second time in 0.5% EmpigenTT containing solution and agitated at ambient temperature on a rocking platform for 5 min before pelleting again.
  • the resulting supernatant was combined with the supernatant from the first EmpigenTT extraction, solid imidazole was added to a final concentration of 10 mM, and the solution was filtered through a 0.2 ⁇ filter (Steriflip, Millipore, Billerica, Mass.). The filtered solution was then passed through a 5 ml Ni Sepharose H isTrap high performance HP column (GE BioScience, Piscataway, N.J.) equilibrated with Lysis Buffer (50 mM sodium phosphate, pH 8, containing 300 mM NaCl, 10 mM imidazole, and 1% (volume/volume) EmpigenTT).
  • Lysis Buffer 50 mM sodium phosphate, pH 8, containing 300 mM NaCl, 10 mM imidazole, and 1% (volume/volume) EmpigenTT.
  • TspB protein was eluted stepwise with Elution Buffer (50 mM sodium phosphate, pH 8, containing 300 mM NaCl, and 1% (volume/volume) EmpigenTT) containing first 125 mM imidazole (14 ml) then 250 mM imidazole (16 ml). 2 ml fractions were collected and analyzed by SDS-PAGE. Fractions containing purified TspB derivatives were combined, dialyzed against 0.9% NaCl and concentrated to 0.2 to 2 mg/ml protein using a YM-10 concentrator (Millipore).
  • Wash Buffer 50 mM sodium phosphate, pH 8, containing 300 mM NaCl, 20 mM imidazole and 1% (volume/volume) EmpigenTT
  • Elution Buffer 50 mM sodium phosphate, pH 8, containing 300 mM NaCl, and 1% (volume/volume) EmpigenTT
  • Flow cytometry experiments were carried out to identify the growing conditions of bacterial cells that might affect the binding of human IgG to the surface of Neisseria meningitidis group W135 (NmW135) strain A22.
  • control cases included no addition and the addition of mouse serum.
  • the results for NmW135 strain A22 are shown in FIG. 1 .
  • the filled histograms show results for the test conditions indicated and the unfilled histograms are the negative controls without additives.
  • Donor 1 who lacks intrinsic serum bactericidal activity against the test strain was added during the binding experiment, no binding or slight binding of IgG was detected (FIG. 1 A,B, respectively).
  • the result shows that there are no antigens expressed by the bacteria under either culture condition that are reactive with antibodies purified from the serum. Again, when the bacteria were cultured in MH ( FIG.
  • FIG. 1E Cells cultured in CDM supplemented with mouse serum (CDM/MoS) showed no binding of mouse IgG ( FIG. 1E ). Also, if the bacteria were cultured in the presence of MoS and purified human IgG was added, no binding of human IgG was observed ( FIG. 1F ). The result shows that factors in human serum eliciting the expression of Ig binding activity are not present in mouse serum. However, if the bacteria were cultured in the presence of human serum and then incubated with mouse serum, binding of mouse IgG was observed but the fluorescence is weaker ( FIG. 1G ). Thus, the Ig binding activity is not entirely specific for human IgG but requires human serum factors to trigger expression of Ig binding activity.
  • the bacteria were grown in CDMHuS supplemented with purified human Fc fragments (prepared from polyclonal human IgG) modified with a fluorescent tag (HuFc-FITC, obtained from Jackson ImmunoResearch Laboratories, West Grove, Pa.). As shown in FIG. 1H , the bacteria bind to the Fc domain alone.
  • Binding is weaker since the Fc domain is competing with intact human IgG present in the culture media or may be weaker because the Fc domain does not represent the entire epitope, but it is clearly increased relative to the negative control of bacteria grown in CDM without dHuS (open histogram) in the presence of HuFc-FITC. Since the Fc fragment lacks a combining site, the observed binding cannot be the result of antibody binding to an antigen but rather an antigen binding the Fc domain. Further, evidence for an Ig binding is provided below in Example 2 where the protein is identified and it is shown that a recombinant subdomain (that is, the IGB domain described above) of the protein binds human and mouse IgA, IgG and IgM.
  • FIG. 2A shows the results of binding experiments using dHuS from two donors (Donor 1 and Donor 3) and human IgG binding by genetically and antigenically diverse group B (NMB) and C (4243) strains in addition to group W135 strains A22, M9262, and 4383 ( FIG. 2B ).
  • NMB genetically and antigenically diverse group B
  • C (4243) strains in addition to group W135 strains A22, M9262, and 4383
  • the result shows that the HuS-dependent expression of the IgG binding protein is not unique to a specific donor and that IgG binding protein expression is not limited to strain A22. Given the genetic and antigenic diversity of the strains and the unlikely exposure of the donors and especially the mice to invasive Nm strains, it is unlikely that the donor sera contain antibodies reactive with a common antigen that is only expressed when the bacteria are cultured in the presence of human serum.
  • the cell pellets were washed (resuspended in buffer and centrifuged) two times in 10 mL of filtered (Steriflip, Millipore) PBS buffer. After the final wash, the cells in 2 mL aliquots were pelleted and frozen on dry ice.
  • the supernatants were transferred to microfuge tubes (1 mL per immunoprecipitation) containing Protein A or G Sepharose CL-4B beads (Sigma) (Protein A: 3-4 mg beads preswollen in 50 ⁇ L of PBS, Protein G: used 50 ⁇ L of beads washed 3 times in PBS). The mixture of sample and beads were incubated overnight with rocking at 4° C. The beads were washed 5 times with solubilization buffer with 2 minute centrifugation to pellet the beads after each wash. After the final wash, the beads were suspended in 75 ⁇ L of sample buffer without 2-mercaptoethanol, and incubated at 100° C. for 5 minutes to release bound immunoglobulin and proteins.
  • the samples were centrifuged as above for 3 minutes to pellet the beads and the supernatant was transferred to a new tube. 2 ⁇ L of 2-mercaptoethanol was added and the supernatant was heated to 100° C. for 5 minutes.
  • the proteins eluted from the beads were resolved on 4%-12% SDS-PAGE gels (Novex precast gels from Invitrogen), which were stained with GelCode Blue (Pierce Chemical Co., Rockford, Ill.) following the manufacturers instructions and the bands in the gel were detected using a LI-COR Odyssey IR imager (Lincoln, Nebr.) as shown in FIG. 3 .
  • the buffer solution and tryptic peptides extracted from in the gel pieces with 0.1% (volume/volume) trifluoroacetic acid (TFA) in 50% (volume/volume) acetonitrile were combined. After drying the eluted peptide solution in a spin-vac (Savant, Thermo-Fisher), the residue was resuspended in 4 ⁇ l of 0.1% TFA/50% acetonitrile. 1 ⁇ l of this solution was added to 3 ⁇ l of 0.1% TFA/50% acetonitrile saturated with ⁇ -cyano-4-hydroxycinnamic acid (Bruker Daltonics, Fremont, Calif.).
  • the number of genes encoding putative TspB/Orf6 proteins ranged from 1 partial sequence (lactamica strain ATCC 23970) to 4 full length sequences (meningococcal group B MC58 group C FAM18 and N. gonorrhoeae strain FA1090). When the translated protein sequences from group A, B, and C strains were compared, a pattern emerged.
  • variable amino terminal sequences are potentially functional leader peptide sequences that direct the nascent protein to be transported out of the cell.
  • the variable sequences are generally hydrophobic membrane spanning segments that may not have to have the same sequence to be functionally equivalent.
  • conserved internal domains there is a typical globular domain that is made up mainly of beta sheet structure based on secondary structure prediction followed by a proline-rich segment. Proline-rich segments are known to adopt extended helical structures (Butcher D J et al. 1996 Biochemistry 35:698-703).
  • TspB/Orf6 genes encode a protein that can be exported to the bacterial surface.
  • the protein contains a highly variable N-terminal domain followed by globular domain extending out from the membrane at the end of the proline-rich helical segment that is anchored to the membrane by a variable membrane spanning segment.
  • the globular and/or proline-rich segments have some functional activity since they are highly conserved while the variable N-terminal domain serves as a dispensable “disguise” for subverting antibody responses.
  • a vaccine designed to elicit antibodies targeting TspB/Orf6 may contain only one or both conserved domains.
  • TspB-IGB The conserved globular domain (CR) was cloned as described below (referred to herein as TspB-IGB).
  • Genomic DNA was prepared from strain MC58 using a Qiagen genomic DNA isolation kit (Qiagen, Valencia, Calif.) following the manufacturers instructions.
  • DNA encoding TspB-IGB was amplified by PCR using the following primers: 5′-CATGGATCCATCAGTTTCCCG-3′ (SEQ ID NO: 49) and 5′-CATAAGCTTTGCTTCCGCGCTTC-3′ (SEQ ID NO: 50).
  • PCR consisted of 30 cycles of denaturation at 95° C., annealing at 50° C., and elongation at 72° C.
  • the PCR fragment was purified using a QIAquick PCR purification kit following the manufacturers instructions and ligated into plasmid pCR2.1 (Invitrogen) using T4 DNA ligase (New England Biolabs, Ipswich, Mass.) overnight at 20° C.
  • the ligation reaction products were used to transform E. coli strain BL21(DE3) (Invitrogen). The transformed E.
  • Plasmid was prepared from white colonies using a Qiagen mini-plasmid prep kit and the presence of the TspB-IGB DNA fragment was confirmed as described below.
  • the plasmid was first digested with Eco RI (New England Biolabs) to confirm the size of the inert. Fragment used in cloning was treated with the restriction endonucleases Hind III followed by Bam HI (New England Biolabs). The resulting fragment was isolated by electrophoresis in a 1% (weight/volume) agarose (Promega, Madison, Wis.) gel containing 0.5 ⁇ g/ml ethidium bromide in TBE buffer (Gibco). The band corresponding to the expected size of the TspB-IGB DNA fragment was isolated by excision from the gel and purified using a Qiagen QIAquick Gel Extraction kit.
  • the Hind III/BamHI TspB-IGB DNA fragment was ligated as before into expression plasmids pQE31 (Qiagen) and pET21b(+) (Novagen now EMD, Gibbstown, N.J.) that were previously digested with the same restriction endonucleases, treated with shrimp alkaline phosphatase (USB, Cleveland, Ohio) and purified by agarose gel electrophoresis as described above. DNA products from the ligation reaction was used to transform strain BL21(DE3), which was plated on LB agar/ampicillin plates as described above. Plasmid DNA was prepared from transformants and the presence of the correct insert was confirmed by restriction endonuclease digestion and DNA sequencing.
  • the plasmid pQE31 allowed for a high level of intracellular expression of TspB-IGB with a His tag (that is a segment containing six histidine residues) at the amino terminus.
  • expression from plasmid pET22b(+) would incorporate a His tag at the carboxyl terminus of TspB-IGB and a pelB leader peptide at the amino terminus of TspB-IGB, which directs the protein for secretion into the periplasm.
  • the advantage of plasmid pQE31 includes very high levels of protein expression while plasmid pET22b(+) allows for native folding in the extracellular periplasmic space.
  • the TspB-IGB containing plasmid constructs were transformed into E. coli strain BL21(DE3) (Invitrogen). Individual colonies were grown overnight in 50 ml of LB media containing 100 ⁇ g/ml ampicillin at 37° C. in a shaking incubator. The next day, the overnight cultures were used to inoculate 500 ml of 2 ⁇ YT media at 37° C. in a shaking incubator. When the OD 620nm was between 0.5 and 1 for cells containing the pQE31-TspB-IGB construct, IPTG (Sigma) was added to a final concentration of 1 mM and amplicillin to 200 ⁇ g/ml.
  • the culture was continued for 3 hrs and the cells harvested by centrifugation at 8000 ⁇ g for 20 minutes, the supernatant was discarded and the cell pellet frozen at ⁇ 80° C. until processed as described below.
  • the culture of BL21(DE3) containing the pET22b(+)-TspB-IGB plasmid was cooled to 22° C. when the OD 620nm was approximately 0.6.
  • IPTG and ampicillin were added to 1 mM and 200 ⁇ g/ml final concentrations, respectively, and the culture was continued at 22° C. overnight. The cells were recovered by centrifugation and frozen until used as described below.
  • NmB strain MC58 has three TspB genes: NMB1548, NMB1628, and NMB1747.
  • the NMB1628 and NMB1747 TspB genes encode polypeptides having identical amino acid sequences. Sequence derived from NMB1628 was cloned in the pQE vector (with an N-terminal HisTag) and the sequences derived from NMB1548 were cloned into both the pQE vector and the pET vector (with a C-terminal HisTag).
  • NMB1628 constant region and proline repeat referred to herein as IGBPro
  • full length NMB1628 in both pQE and pET vectors See details below.
  • TspB-IGBpro A DNA fragment spanning the conserved globular domain followed by the conserved proline-rich repeat of NMB1628 was cloned as described below (referred to herein as TspB-IGBpro). Genomic DNA was prepared from strain MC58 using the Qiagen genomic DNA isolation kit (Qiagen, Valencia, Calif.) following the manufacturers instructions. DNA encoding TspB-IGBpro (NMB1628) was amplified by PCR using the following primers: 5′-CATGGATCCATCAGTTTCCCGCGCCGCCGTCTT-3′ (SEQ ID NO: 51) and 5′-CATAAGCTTGTTCTCAAAGCTGAACGCG-3′ (SEQ ID NO: 52).
  • PCR consisted of 5 cycles of denaturation at 95° C., annealing at 48° C., and elongation at 72° C., followed by 25 cycles of denaturation at 95° C., annealing at 52° C., and elongation at 72° C., with an Idaho Technology (Salt Lake City, Utah) RapidCycle thermocycler, employing the Taq DNA polymerase, nucleotides and reaction buffers from New England Biolabs (Ipswich, Mass.).
  • the PCR fragment was purified using a QIAquick PCR purification kit (Qiagen) following the manufacturers instructions.
  • the PCR products were ligated into plasmid pCR2.1 (Invitrogen) and ligation reaction products were used to transform E. coli strain TOP 10 (Invitrogen).
  • the transformed E. coli cells were plated on LB agar plates containing 100 ⁇ g/ml ampicillin (Sigma) and X-Gal (bromo-chloro-indolyl-galactopyranoside, Gold Biotechnology).
  • the insert-carrying plasmid pCR2.1 was digested with restriction endonucleases Bam HI and Hind III (New England Biolabs). The resulting fragment was isolated by electrophoresis in a 1.5% (weight/volume) agarose (Promega, Madison, Wis.) gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA). After electrophoresis DNA bands were visualized by submerging the agarose gel in 0.5% ethidium bromide solution (Gibco). The band corresponding to the expected size of the TspB-IGBpro DNA fragment was isolated by excision from the gel and purification using a Qiagen QIAquick Gel Extraction kit.
  • the Bam HI/Hind III TspB-IGBpro DNA fragment was ligated into expression plasmid pQE31 (Qiagen) that was previously digested with the same restriction endonucleases and treated with shrimp alkaline phosphatase (USB, Cleveland, Ohio).
  • the ligation reaction was then used to transform strain BL21(DE3) (Invitrogen), which was plated on LB agar/ampicillin plates as described above.
  • DNA was then prepared from chosen transformants and the correct sequence encoding TspB-IGBpro was verified by DNA sequencing.
  • TspB-FL The full length open reading frame (start to stop codon) of NMB1628 was cloned as described below (referred to herein as TspB-FL). Genomic DNA was prepared from strain MC58 using the Qiagen genomic DNA isolation kit (Qiagen, Valencia, Calif.) following the manufacturers instructions.
  • DNA encoding TspB-FL was amplified by PCR using primer pair 5′-AGCATATGTTGGGGATGTTTTCGGT-3′ (SEQ ID NO: 55) and 5′-AGAAGCTTGACTTCACGAGATACTGTGC-3′ (SEQ ID NO: 56) for cloning into plasmid pET22b(+), and 5′-ACGGATCCTTGGGGATGTTTTCGGTTAA-3′ (SEQ ID NO: 57) and 5′-AGAAGCTTCTAGACTTCACGAGATACTG-3′ (SEQ ID NO: 58) for cloning into plasmid pQE30.
  • PCR for both amplification reactions consisted of 5 cycles of denaturation at 95° C., annealing at 48° C., and elongation at 72° C., followed by 25 cycles of denaturation at 95° C., annealing at 52° C., and elongation at 72° C., with an Idaho Technology (Salt Lake City, Utah) RapidCycle thermocycler, employing the Taq DNA polymerase, nucleotides and reaction buffers from New England Biolabs (Ipswich, Mass.).
  • the PCR fragments were purified using a QIAquick PCR purification kit (Qiagen) following the manufacturers instructions.
  • the PCR products were ligated into plasmid pCR2.1 (Invitrogen) and ligation reaction products were used to transform E. coli strain TOP 10 (Invitrogen).
  • the transformed E. coli cells were plated on LB agar plates containing 100 ⁇ g/ml ampicillin (Sigma) and X-Gal (Gold Biotechnology). Plasmids were prepared from white colonies that generated PCR products during colony PCR with the primers used for initial amplification of TspB-FL fragments using a Qiagen mini-plasmid prep kit. Correct TspB-FL insert sequences were confirmed via DNA sequencing.
  • the plasmids were digested with the restriction endonucleases Nde I and Hind III, or Bam HI and Hind III (New England Biolabs) to liberate the fragments used for cloning into vectors pET22b(+) and pQE30, respectively.
  • the resulting fragments were isolated by electrophoresis in a 1.5% (weight/volume) agarose (Promega, Madison, Wis.) gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA). After electrophoresis DNA bands were visualized by submerging the agarose gel in 0.5% ethidium bromide solution (Gibco).
  • the bands corresponding to the expected sizes of the TspB-FL DNA fragment were isolated by excision from the gel and purified using a Qiagen QIAquick Gel Extraction kit.
  • the Nde I/Hind III and Bam HI/Hind III TspB-FL DNA fragments were ligated into the expression plasmids pET22b(+) and pQE30 (Qiagen), respectively, which were previously digested with the same restriction endonucleases and treated with shrimp alkaline phosphatase (USB, Cleveland, Ohio).
  • DNA products from the ligation reactions were used to transform E. coli strain TOP 10 F′ cells (Invitrogen), which were plated on LB agar/ampicillin plates as described above. Plasmids were prepared from transformants that generated PCR products during colony PCR, as described above, using a Qiagen mini-plasmid prep kit. Correct TspB-FL insert sequences were again confirmed via DNA sequencing.
  • TspB-IGB encoded by NMA0776 and NMA1797 were cloned as described above for the TspB-IGBpro encoded by NMB1628 in MC58.
  • Genomic DNA was prepared from strain Z2491 using a Qiagen genomic DNA isolation kit (Qiagen, Valencia, Calif.) following the manufacturers instructions.
  • DNA encoding TspB-IGB of NMA0776 was amplified by PCR using the following primers: 5′-CATGGATCCATCAGTATCCCGCGCCG-3′ (SEQ ID NO: 59) and 5′-CATAAGCTTTGCTTCTGCGCTTCCG-3′ (SEQ ID NO: 60), while DNA encoding TspB-IGB of NMA1797 was amplified by PCR using the following primers: 5′-CATGGATCCATCAGTTTCCCGCGCCG-3′ (SEQ ID NO: 61) and 5′-CATAAGCTTTGCTTCCGCGCTTC-3′ (SEQ ID NO: 62).
  • PCR for both amplification reactions consisted of 5 cycles of denaturation at 95° C., annealing at 48° C., and elongation at 72° C., followed by 25 cycles of denaturation at 95° C., annealing at 52° C., and elongation at 72° C., with an Idaho Technology (Salt Lake City, Utah) RapidCycle thermocycler, employing the Taq DNA polymerase, nucleotides and reaction buffers from New England Biolabs (Ipswich, Mass.).
  • the PCR fragments were purified using a QIAquick PCR purification kit (Qiagen) following the manufacturers instructions.
  • the PCR products were ligated into plasmid pCR2.1 (Invitrogen) and ligation reaction products were used to transform E. coli strain TOP 10 (Invitrogen).
  • the transformed E. coli were plated on LB agar plates containing 100 ⁇ g/ml ampicillin (Sigma) and X-Gal (Gold Biotechnology). Plasmids were prepared from white colonies that generated PCR products during colony PCR with the primers used for initial amplification of TspB-IGB encoded by NMA0776 and NMA1797 using a Qiagen mini-plasmid prep kit. Correct TspB-IGB insert sequences were confirmed via DNA sequencing.
  • Plasmids carrying both TspB-IGB insertions sequences were digested with restriction endonucleases Bam HI and Hind III (New England Biolabs) to liberate the two fragments used in cloning.
  • the resulting fragments were isolated by electrophoresis in a 1.5% (weight/volume) agarose (Promega, Madison, Wis.) gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA). After electrophoresis DNA bands were visualized by submerging the agarose gel in 0.5% ethidium bromide solution (Gibco).
  • the bands corresponding to the expected sizes of the TspB-IGB DNA fragments were isolated by excision from the gel and purified using a Qiagen QIAquick Gel Extraction kit.
  • the Hind III/BamHI TspB-IGB DNA fragments were ligated into expression plasmid pQE31 (Qiagen) that was previously digested with the same restriction endonucleases and treated with shrimp alkaline phosphatase (USB, Cleveland, Ohio).
  • the ligation reactions were used to transform E. coli strain TOP 10 F′ (Invitrogen), which was plated on LB agar/ampicillin plates as described above.
  • Plasmid DNA was prepared from colonies that generated PCR products during colony PCR with the primers used for initial amplification of the two TspB-IGB fragments using a Qiagen mini-plasmid prep kit. Correct TspB-IGB insert sequences were confirmed via DNA sequencing.
  • TspB-IGB proteins Purification of the TspB-IGB proteins was performed using a 5 ml Ni Sepharose HisTrap high performance HP column (GE BioScience, Piscataway, N.J.) under denaturing conditions for pQE31-TspB-IGB expression plasmid or native conditions for pET22b(+)-TspB-IGB expression plasmid, as described in the Qiaexpressionist Handbook 5 th Edition (2003, Qiagen). Fractions from the column were analyzed by SDS-PAGE using precast gels from Invitrogen followed by staining with SimplyBlue SafeStain (Invitrogen) and documented by scanning the gel with on Odyssey IR scanner (LI-COR).
  • Fractions containing TspB-IGB purified under denaturing conditions were combined (6 ml total) diluted to 30 ml in 100 mM NaH 2 PO 4 buffer, pH 8.0, containing 10 mM Tris.HCl, and 8M urea and dialyzed (1 kDa cutoff Spectropor membrane, Fisher) in PBS buffer (1 L total) at ambient temperature by adding 25 ml of PBS buffer at a time over the course of 1 day.
  • TspB protein was dialyzed in 4 L of 2 mM histidine, pH 6, containing 0.002% Tween 20 and 24 mM sucrose and lyophilized TspB-IGB purified under native conditions (pET22b(+)-TspB-IGB construct) was dialyzed in 4 L of 2 mM histidine, pH 6, containing 0.002% Tween 20 and 24 mM sucrose and lyophilized
  • FIG. 6 the purification products expressed from either the pQE31 or the pET22b(+) plasmids demonstrated that both methods described above can produce TspB-IGB polypeptides of high purity.
  • FIG. 6 lane A shows TspB-IGB expressed from pQE31 and purified under denaturing conditions and refolded.
  • FIG. 6 lane B shows TspB-IGB expressed from pET21b(+) purified under native conditions from the periplasm.
  • TspB-IGB polypeptides produced and purified under denaturing conditions then refolded or purified under native conditions were evaluated by solid phase ELISA assay for their abilities to bind to IgG from various sources.
  • TspB-IGB 100 ⁇ l/well at a concentration of 10 ⁇ g/ml in PBS
  • microtiter plates Immulon 2; Dynatech Laboratories, Inc., Chantilly, Va.
  • the plates were blocked with 250 ⁇ l of blocking buffer (PBS containing 1% bovine serum albumin and 0.1% sodium azide, pH 7.4) for 30 to 60 minutes at room temperature.
  • the wells were washed five times with PBS and were incubated for one hour at ambient temperature with 100 ⁇ l/well of alkaline phosphatase-conjugated anti-human (Jackson ImmunoResearch, West Grove, Pa.) or anti-mouse polyclonal antibody (IgA+IgG+IgM; Zymed, South San Francisco, Calif.) diluted 1:3000 in blocking buffer.
  • the plates were then washed with PBS, and 100 ⁇ l of freshly prepared substrate (p-Nitrophenyl phosphate; Sigma) diluted to 1 mg/ml in substrate buffer (50 mM Na 2 CO 3 , 1 mM MgCl 2 , pH 9.8) was added to each well. Absorbance values at 405 nm were measured after approximately 60 minutes.
  • the IgG antibody concentration in each sample was determined by capture ELISA (Southern Biotech for mouse and Immunological Consultants for human). Table 1 below summarizes the binding data.
  • Antibody concentrations in stock solutions were measured by capture ELISA except for Ig standards, which were provided by the manufacturer.
  • DA2 was produced in CHO cells and SEAM 12 in a hybridoma cell line.
  • Negative control was BSA, which was the same as secondary only at data point dilution.
  • TspB-IGB contains at least a portion of the IgG binding functional activity of full length TspB and that TspB-IGB can bind both human and mouse IgG.
  • human Ig concentrations of Ig giving equivalent ELISA readings were 5-10-fold lower for human Ig.
  • fluorescence of cells in the presence of mouse Ig was lower than in the presence of human Ig (compare FIG. 1 , D and G).
  • the reasons for the apparent lack of reactivity with some of the commercially obtained isotype standards is unclear but may have to do with differences in Fc glycoforms that occur in myelomas (Farooq M et al.
  • TspB-IGB a domain of the full length TspB protein that are absent in TspB-IGB may contribute to binding.
  • the proline-rich segment may promote the formation of multimers that would be expected to exhibit greater avidity than monomeric forms.
  • TspB IGB proteins encoded by NMB1628 and NMB1548, expressed from the pQE plasmid with a HisTag at the amino terminus, and purified under native conditions were adsorbed (100 ⁇ l of 10 ⁇ g/ml) to the wells of a microtiter plate as described above. After blocking with Blocking Buffer, serial dilutions of purified human IgG in Blocking Buffer were added to the wells and left at 4° C. overnight. The plates were washed and bound IgG was detected as described above.
  • FIG. 11 shows that TspB IGB encoded by NMB1628 bound strongly to purified human IgG while TspB IGB encoded by NMB1548 bound human IgG poorly.
  • TspB IGB encoded by NMB1628 FIG. 12 , panel A
  • TspB IGB encoded by NMB1548 FIG. 12 , panel B
  • the ability of TspB IGB derivatives to form polymeric structures can be associated with the ability to bind human IgG.
  • FIG. 13 shows the ability of several N. meningitidis strains from different capsular groups to bind human IgG from two different donor sera (Donor 1 and Donor 2) as determined by flow cytometry (Example 1). Some of the strains (e.g. NmC strain 4243) bound to IgG strongly while other (e.g. NmW135 stain A22) had little or no binding. Strains A22, NMB, and Z1092, which showed little or no IgG binding were subjected to selection in the presence of human complement as follows.
  • Bacterial colonies growing on chocolate agar were used to inoculate a 5 ml culture of Catlin 6 media containing 5% (volume/volume) heat inactivated IgG depleted human serum (CDMHuS) (prepared as described in Materials and Methods) to an OD 620nm of 0.18 and were grown at 37° C. to an OD 620nm of 0.72.
  • the culture was centrifuged to collect the bacteria, which were suspended in the same volume of culture media.
  • a portion of bacterial solution was diluted 1:80,000 in 1 ml of CDM containing 20% (volume/volume) human complement. The bacteria were grown overnight at 37° C.
  • the bacteria were diluted to an OD 620nm of 0.18 in 5 ml of CDMHuS and grown again to an OD 620nm of 0.72. The procedure was repeated two times and the bacteria obtained after each round of overnight selection in 20% human complement were analyzed for IgG binding by flow cytometry.
  • the fraction of cells that were positive for IgG binding increased with each round of selection.
  • IgG binding to the strain before selection ( FIG. 13 ) is shown in the unfilled histogram.
  • Expression of IgG binding activity could be variable in bacteria that were not grown in the presence of human serum factors. However, IgG binding was restored by repeated culture in human serum containing active complement.
  • Bacteria used for measuring binding and to be used for bactericidal activity (BCA) of anti-TspB-IGB antibodies described in the Examples below were selected using the methods described in this Example.
  • the bacteria were stored for future use by suspending the bacteria grown in 5 ml of CDMHuS in 200 ⁇ l of whole human serum then freezing 20 ⁇ l aliquots on dry ice. The bacterial aliquots were stored at ⁇ 80° C. until used.
  • Antisera were collected from immunized mice as described above in the Materials and Methods section. Mice was immunized with the following:
  • NMB1628IGB expressed in pQE purified under denaturing and native conditions rfTspB1628IGB
  • NMB1628 IGBPro produced from pQE and purified under native conditions (nTspB1628IGB);
  • NMB1548 produced from pET and purified under native conditions (nTspB1548IGB).
  • the ability of the TspB-IGB-based vaccines to elicit antibodies that bind to Neisseria was tested by flow cytometry.
  • the Neisseria strains were grown to an O.D. 620 of ⁇ 0.6 in CDM or CDMHuS so that cells were induced to express TspB/Orf6 can be compared to uninduced cells.
  • the cells were pelleted, washed, and resuspended in 80% of the original volume in blocking buffer.
  • the bacterial suspension was then added to the reaction mixture such that the final concentrations will be 50% bacterial suspension, 10% antiserum, and 40% blocking buffer.
  • the mixture was incubated at 4° C. for 2 hr with periodic gentle agitation.
  • the cells were pelleted by centrifugation and resuspended in 100 ⁇ l of a 1:300 dilution in blocking buffer of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse secondary antibodies.
  • FITC-conjugated antibodies against IgG(H+L) F(ab′) 2 and IgM Jackson ImmunoResearch, West Grove, Pa.
  • IgG1, IgG2a, IgG2b, and IgG3 Bethyl Laboratories, Montgomery, Tex.
  • the cells were pelleted and resuspended in 450 ⁇ l of PBS containing 0.5% formaldehyde (weight/volume), freshly made and filtered. The samples were then analyzed by flow cytometry (BD FACSCalibur System, BD Biosciences, San Jose, Calif.).
  • FIG. 15 shows flow cytometry experiments of anti-1628IGB binding to NmB strain MC58 ( FIG. 15 , panel A) or to NmA strain Z2491 ( FIG. 15 , panel B).
  • Controls included no added antiserum or adjuvant only control serum.
  • graphs shifted to the right, which corresponded to increased fluorescence of the anti-1628IGB.
  • antibodies elicited by the vaccine bound strongly to both NmA and NmB strains.
  • panels A and B show binding of human IgG to each strain.
  • Levels of binding of human IgG to the bacteria were decreased in the presence of antisera to rfTspB1628IGB, suggesting that the antisera inhibited binding of human IgG to the bacteria.
  • FIG. 16 shows a similar binding experiment with NmB strain MC58 in which the binding of antibodies elicited by the following immunogens were compared
  • the cells were grown in CDM+5% Cohn FIV+1% purified human IgG and a 1 to 200 dilution of the indicated antiserum. After the cells reached an OD620 nm of 0.6, they were divided into two equal fractions. One fraction was treated with the anti-mouse IgG-FITC and the other with the anti-human IgG-FITC secondary antibodies to detect binding of the respective IgG. The results show that rfTspB1628IGB elicted IgG antibodies that bind the most strongly to NmB strain MC58.
  • TspB particularly the IGB domain, was found to be the protein expressed by Nm strains that mediates human IgG binding activity. Therefore, antibodies elicited by a TspBIGB-based vaccine can block human IgG binding, which is an important mechanism for survival of Nm strains in human blood.
  • the ability of antibodies elicited by immunization with TspB IGB vaccine to activate deposition of human complement components on Neisseria meningitidis bacteria representative of groups B, C, X, Y, and W135 bacteria can be determined as described below.
  • the TspB-IGB vaccine used to elicit antibodies may contain TspB-IGB polypeptide combined with any adjuvant (e.g. aluminum hydroxide, aluminum phosphate, MF59, Freund's adjuvant, etc.).
  • adjuvant e.g. aluminum hydroxide, aluminum phosphate, MF59, Freund's adjuvant, etc.
  • Complement activation by the antisera of the vaccine may be determined as described by Welsch et al (Welsch et al, 2003 J. Infec. Dis. 188:1730). Briefly, the Neisseria cells are grown to an OD 620 of ⁇ 0.6 in CDM/dHuS.
  • the cells are pelleted, washed with Dulbecco's PBS (Invitrogen, Carlsbad, Calif.) containing 1% (weight/volume) BSA (Sigma-Aldrich) (D-BSA), and resuspended in half the volume D-BSA of the original growth media.
  • the bacteria (30 ⁇ l) are combined with the pooled antisera from mice immunized with either 2 ⁇ g or 10 ⁇ g diluted to 1:20 or 1:200 in D-BSA.
  • Human complement from a donor tested for the absence of antibodies to Neisseria cells is added to a concentration of 5% (volume/volume) in a total volume of 200 ⁇ l. The reaction is allowed to proceed for 30 minutes at ambient temperature with occasional agitation.
  • the cells are pelleted and washed with 200 ⁇ l of D-BSA and FITC-conjugated sheep anti-human C3c antibody (BioDesign International, Saco, Me.) is added in D-BSA. After 30 minutes incubation on ice with occasional agitation, the cells are pelleted, resuspended in sterile filtered PBS buffer containing 0.5% (weight/volume) formaldehyde and analyzed by flow cytometery (BD FACSCalibur System, BD Biosciences, San Jose, Calif.).
  • Deposition of complement components on the cell surface increases the fluorescence of the cells and may be indicated by the shift in fluorescence peak to the right of a graph.
  • the antibody activation may be indicated by the lack of fluorescence of cells alone with active complement or antisera with heat-inactivated complement.
  • the bacteria from flow cytometry are analyzed further by microscopy using a Zeiss Axioplan (Carl Zeiss, Inc.) fluorescence microscope. Activation of complement factor deposition on the cell surface of NmB bacteria is correlated with protection against disease caused by Neisseria meningitidis (Welsch et al. 2003 J. Infec. Dis. 188:1730).
  • the ability of anti-TspB IGB antiserum to mediate bacteriolysis in the presence of human complement can be measured by the serum bactericidal assay (SBA).
  • SBA serum bactericidal assay
  • the bactericidal assay is performed as previously described (Moe G. R. et al. 2002 Infect Immun 70: 6021-31) using mid-log phase bacteria grown overnight on chocolate agar plates then cultured in CDM/dHuS, or for comparison to cells not induced to express TspB IGB, Mueller Hinton media broth supplemented with 0.25% glucose will be used. Cells taken from the chocolate agar plates will be diluted in media to OD 620nm of ⁇ 0.18 to start the culture then grown to an OD 620nm of ⁇ 0.62.
  • the bacteria will be pelleted and then washed in Dulbeccos buffered saline with Ca+ and Mg+ (DBS; Mediatech. Manassas Va.), with or without 1% BSA (Equitech).
  • DBS Dulbeccos buffered saline with Ca+ and Mg+
  • BSA BSA
  • the human complement sources used in each assay are evaluated for intrinsic SBA against test strains in the presence of 60% complement.
  • the final reaction mixture contains different dilutions of test sera, and 20% (volume/volume) human complement.
  • the buffer is Dulbecco's phosphate buffered saline (Sigma-Aldrich) containing 0.9 mM CaCl2 ⁇ 2 H2O, 0.5 mM MgCl2 ⁇ 6 H2O and 1% (weight/volume) BSA.
  • the complement source is human serum from a healthy adult with no detectable intrinsic bactericidal activity (Granoff et al. J 1998 Immunol 160:5028-36; Welsch et al. 2003 J. Infec. Dis. 188:1730).
  • Serum bactericidal titers are defined as the serum dilution resulting in a 50% decrease in CFU per ml after 60 minutes of incubation of bacteria in the reaction mixture, as compared with control CFU per ml at time 0.
  • bacteria incubated with the negative control antibody and complement show a 150 to 200% increase in CFU/mL during the 60 minutes of incubation.
  • Anti-TspB-IGB antisera can be tested for passive protection against bacteremia as described below. The results of this experiment can determine the ability of a vaccine based on TspB-IGB to protect against invasive disease in humans.
  • the pups are anesthetized with isoflurane and blood obtained by cardiac puncture using a heparanized needle.
  • the animals are euthanized by CO 2 anoxia, and 100 ⁇ l, 10 ⁇ l, and 1 ⁇ l of the blood will be plated on chocolate agar (Remel). After incubating the plates overnight at 37° C., 5% CO 2 the colonies will be counted.
  • Antisera tested are pooled antisera from CD1 mice immunized with TspB-IGB-based vaccines as described above) and the test bacteria (approximately 1000 CFU of a Neisseria strain freshly grown in CDM/dHuS media as described above) are combined in freshly obtained human blood from a donor who lacks antibodies to the test strain in sterile glass vials and prepared and tested as described below.
  • Fresh blood from a healthy adult is obtained using a syringe containing recombinant hirudin (lepirudin, 27.8 ⁇ g/ml final concentration) as the anti-coagulant.
  • the blood donor may be the same person whose serum was used for complement to measure serum bactericidal activity described above.
  • To each well of the microtiter plate 65 ⁇ l of blood are added, 25 ⁇ l of test sera, and 10 ⁇ l of PBS buffer containing 15% heated-complement and 1% Bovine Serum Albumin, (Equitech) and approximately 4000 CFU.
  • the positive controls may include a relevant anti-PorA mAb (NIBSC, P1.4 or P1.12) and human serum samples with high, medium or low opsonophagocytic killing activity (Plested and Granoff 2008 Clin Vaccine Immunol 15: 799-804) and serum bactericidal activity against the respective test strains.
  • Negative control sera may be heat-inactivated serum from the complement donor or buffer alone.
  • the microtiter plates are incubated for 90 minutes at 37° C. in 5% CO 2 with agitation on a MS 3 digital minishaker (IKA, Wilmington, N.C.) at 500 oscillations per minute to completely mix the components. Samples are removed from the wells and quantitative cultures performed on chocolate agar plates, which are incubated at 37° C. in 5% CO 2 .
  • a positive passive protection activity may be defined as a serum giving ⁇ 0.5 log 10 decrease in CFU/ml, as compared to that at time 0 (i.e., about 70% decrease in CFU/ml).

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Gene ID: NMA1173 "NMA1173 cell-surface protein [ Neisseria meningitidis Z2491 ]" dated Dec. 22, 2012.
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Gene ID: NMB1628 "NMB1628 tspB protein [ Neisseria meningitidis MC58 ]" dated Dec. 5, 2011.
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AU2010315115B2 (en) 2015-01-29

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